引导骨再生壳聚糖—胶原可吸收膜的研制及其物理性能、生物相容性的研究

发布时间：2018-05-04 03:23

本文选题：壳聚糖 + 胶原　；参考：《浙江大学》2012年硕士论文

【摘要】：目的：引导骨再生技术(Guided Bone Regeneration,GBR)的出现,为局部牙槽骨缺损的患者行种植义齿修复提供了可能。目前,GBR已成为一种常规的牙种植辅助技术,可改善缺牙区骨组织的质量和数量,以满足种植体的植入条件。而屏障膜在GBR技术中起着重要作用。本研究将壳聚糖和胶原以不同比例混合,通过机械性能测试、体外细胞增殖实验和动物体内埋植实验对其进行物理性能、生物相容性及降解性研究,以期将自主研发的壳聚糖-胶原可吸收膜应用于牙种植引导骨再生技术。方法：(1)将2%的低分子量壳聚糖(MW50,000～190,000)冰乙酸溶液与0.3%的胶原水溶液以体积比2：1、1：1、1：2(定义为A,B,C组)混合,加入1%的NaOH溶液调节混合液至pH呈中性,混合液经4℃静置过夜除气泡、-20℃冷冻24h和-80℃预冻12h后,采用冻干法制备A、B、C三组壳聚糖-胶原膜。通过拉伸实验和压汞实验研究膜的物理性能。用扫描电镜观察比较冻干膜与Bio-Gide膜表面形态的差异。 (2)将2%的低分子量壳聚糖冰乙酸溶液(CHS-L)、1%的中分子量壳聚糖(MW190,000～310,000)冰乙酸溶液(CHS-M)及0.1%的胶原(MW300,000)冰乙酸溶液(COL)分别透析至溶液pH呈中性。分别将CHS-L和CHS-M与COL以2：1和4：1的体积比混合,得到四种混合液。混合液经4℃静置过夜除气泡、-20℃冷冻48h和-80℃预冻24h后,采用冻干法制备壳聚糖-胶原膜。将膜裁剪至所需大小后,独立包装EO灭菌。 (3)将灭菌膜片浸入配好的细胞培养液中,膜表面积与培养液比例为6cm2/ml,用膜浸提液和常规细胞培养液培养MC3T3-E1细胞,采用MTT实验评价膜对细胞增殖活性的影响。 (4)将灭菌膜片植入8只新西兰兔的背部皮下组织内,手术后1周、2周、4周、8周各随机处死2只动物,取出剩余膜材料。通过计算膜植入前后的质量差测定膜在动物体内的降解率。结果：(1)低分子量壳聚糖冰乙酸溶液与胶原水溶液混合制备的A(2：1)、B(1：1)、C(1：2)三组冻干膜,以2：1和1：1比例制备的膜成膜效果较好,以1：2比例制备的膜成膜效果较差(不进行物理性能评价)。拉伸实验结果表明,以2：1比例制备的膜机械性能优于1：1比例制备的膜；压汞实验显示,以2：1和1：1比例制备的膜存在广泛的孔隙,孔隙率分别为89.41%和83.92%；通过扫描电镜观察,2：1和1：1比例制备的两组冻干膜孔隙均较Bio-Gide膜大。 (2)2%的低分子量壳聚糖冰乙酸溶液、1%的中分子量壳聚糖冰乙酸溶液和0.1%的胶原冰乙酸溶液分别透析至溶液呈中性后,未见絮状物质析出。壳聚糖溶液可与胶原溶液均匀混合,混合液经真空冻干可制成膜。两种中分子量壳聚糖冻干膜的厚度和密度,均大于低分子量壳聚糖制备的冻干膜。 (3)中分子量壳聚糖制备的两种冻干膜,对小鼠MC3T3-E1细胞增殖无抑制。壳聚糖与胶原体积比为4：1时,其浸提液对MC3T3-El细胞的增殖表现出显著的促进作用,特别是从第4天开始,OD490值明显高于对照组(P0.01)；壳聚糖与胶原体积比为2：1时,其浸提液对MC3T3-E1细胞增殖无显著影响,OD490值仅稍高于对照组(P0.05)。 (4)皮下埋植实验显示,两种中分子量壳聚糖-胶原冻干膜植入动物皮下组织后未见明显的炎症反应。两种壳聚糖-胶原冻干膜在动物体内可降解吸收。8周时,壳聚糖-胶原冻干膜(2：1和4：1)的降解率分别为43.83%和26.45%,但未降解的膜材料已破碎或溶胀呈浆状。结论：(1)壳聚糖和胶原混合制备的冻干膜有较好的物理性能和生物相容性。(2)中分子量壳聚糖(MW190,000-310,000)和胶原的冰乙酸溶液分别透析至中性后以体积比4：1混合,经真空冻干制备的复合可吸收膜,具有良好的成膜性、生物相容性及生物降解性,具备了应用于引导骨再生技术的基本条件。
[Abstract]:Objective: to guide the appearance of Guided Bone Regeneration (GBR) to repair the implant denture for the patients with local alveolar bone defects. At present, GBR has become a conventional dental implant assisted technique to improve the quality and quantity of the bone tissue in the odontic area to meet the implant conditions. And the barrier membrane is in the GBR technique. In this study, the physical properties, biocompatibility and degradability of chitosan and collagen were mixed in different proportions, through mechanical performance testing, in vitro cell proliferation experiments and animal implantation experiments, in order to apply the self developed chitosan collagen absorbable membrane to dental implant guided bone regeneration. Technology.
Methods: (1) the mixture of 2% low molecular weight chitosan (MW50000 ~ 190000) glacial acetic acid and 0.3% collagen solution was mixed with 2:1,1:1,1:2 (defined as A, B, C), and 1% NaOH solution was added to regulate the mixture to pH, and the mixture was statically overnight degassed by 4, and the freeze drying process was adopted after -20 C freezing 24h and -80 C prefreeze. The A, B, C three groups of chitosan collagen membrane were prepared. The physical properties of the membrane were studied by tensile test and mercury pressure test. The surface morphology of freeze-dried film and Bio-Gide film was compared with the scanning electron microscope.
(2) 2% low molecular weight chitosan glacial acetic acid solution (CHS-L), 1% medium molecular weight chitosan (MW190000 ~ 310000) glacial acetic acid solution (CHS-M) and 0.1% collagen (MW300000) glacial acetic acid solution (COL) were dialytic to pH neutrality respectively. The mixture of CHS-L and CHS-M and COL were mixed with 2:1 and 4:1 volume ratio, and mixed solution was obtained. The chitosan collagen membrane was prepared by freeze-drying to prepare the chitosan collagen membrane after freezing at -20 C for 48h and -80 C for 24h at 4 C for overnight defroding, and the membrane was cut to the required size to be sterilized independently by EO.
(3) the membrane surface area and the culture solution were 6cm2/ml, MC3T3-E1 cells were cultured with membrane leaching solution and conventional cell culture solution, and the effect of membrane on cell proliferation activity was evaluated by MTT test.
(4) the sterilization film was implanted into the subcutaneous tissue of the back of 8 New Zealand rabbits. 1 weeks, 2 weeks, 4 weeks and 8 weeks after the operation, 2 animals were killed at random. The residual membrane material was taken out. The degradation rate of the membrane in the animal was measured by calculating the poor quality of the membrane before and after implantation.
Results: (1) A (2:1), B (1:1), C (1:2) three groups of freeze-dried membranes prepared with low molecular weight chitosan glacial acetic acid solution, C (1:2), were prepared by the proportion of 2:1 and 1:1, and the membrane formation effect was poor in 1:2 ratio (no physical ability evaluation). The tensile test results showed that the ratio of 2:1 was prepared in the ratio of 2:1. The membrane mechanical properties are superior to those prepared by the 1:1 ratio. The experiments of mercury injection show that there are extensive pores in the films prepared in the proportion of 2:1 and 1:1, with the porosity of 89.41% and 83.92%, respectively. By scanning electron microscopy, the pores of the two groups of freeze-dried membrane prepared by the proportion of 2:1 and 1:1 are larger than those of the Bio-Gide film.
(2) 2% low molecular weight chitosan glacial acetic acid solution, 1% medium molecular weight chitosan glacial acetic acid solution and 0.1% collagen glacial acetic acid solution were respectively dialytic to neutral, no floc substance was found. Chitosan solution could be mixed with collagen solution evenly and mixed solution could be made into membrane through vacuum freeze drying. Two kinds of molecular weight chitosan lyophilized membrane The thickness and density are all larger than those of freeze-dried membranes prepared by low molecular weight chitosan.
(3) the two lyophilized membranes prepared by medium molecular weight chitosan did not inhibit the proliferation of MC3T3-E1 cells. When the volume ratio of chitosan and collagen was 4:1, the extracts of the chitosan and collagen showed significant promoting effect on the proliferation of MC3T3-El cells, especially from the beginning of fourth days, the OD490 value was obviously higher than that of the opposite group (P0.01), and the volume ratio of chitosan to collagen was 2:1. The extract had no significant effect on the proliferation of MC3T3-E1 cells, and the OD490 value was only slightly higher than that of the control group (P0.05).
(4) subcutaneous implantation experiments showed that two kinds of chitosan collagen lyophilized membrane had no obvious inflammatory reaction after implantation in animal subcutaneous tissue. The degradation rates of chitosan collagen lyophilized membrane (2:1 and 4:1) were 43.83% and 26.45% respectively, but the undegraded membrane materials of two kinds of chitosan collagen lyophilized membrane were degraded for.8 weeks in animals. It has been broken or swelling in the shape of a pulp.
Conclusion: (1) the lyophilized membrane prepared by chitosan and collagen has good physical properties and biocompatibility. (2) the mixture of chitosan (MW190000-310000) and collagen in acetic acid solution, after dialysis to neutral, is mixed with 4:1, and the composite absorbable membrane prepared by vacuum freeze-drying has good film forming and biocompatibility. And biodegradability, which have the basic conditions for guiding bone regeneration technology.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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