本文選題：殼聚糖 + 膠原��； 參考：《浙江大學(xué)》2012年碩士論文

【摘要】：目的：引導(dǎo)骨再生技術(shù)(Guided Bone Regeneration,GBR)的出現(xiàn),為局部牙槽骨缺損的患者行種植義齒修復(fù)提供了可能。目前,GBR已成為一種常規(guī)的牙種植輔助技術(shù),可改善缺牙區(qū)骨組織的質(zhì)量和數(shù)量,以滿足種植體的植入條件。而屏障膜在GBR技術(shù)中起著重要作用。本研究將殼聚糖和膠原以不同比例混合,通過機械性能測試、體外細胞增殖實驗和動物體內(nèi)埋植實驗對其進行物理性能、生物相容性及降解性研究,以期將自主研發(fā)的殼聚糖-膠原可吸收膜應(yīng)用于牙種植引導(dǎo)骨再生技術(shù)。 方法：(1)將2%的低分子量殼聚糖(MW50,000～190,000)冰乙酸溶液與0.3%的膠原水溶液以體積比2：1、1：1、1：2(定義為A,B,C組)混合,加入1%的NaOH溶液調(diào)節(jié)混合液至pH呈中性,混合液經(jīng)4℃靜置過夜除氣泡、-20℃冷凍24h和-80℃預(yù)凍12h后,采用凍干法制備A、B、C三組殼聚糖-膠原膜。通過拉伸實驗和壓汞實驗研究膜的物理性能。用掃描電鏡觀察比較凍干膜與Bio-Gide膜表面形態(tài)的差異。 (2)將2%的低分子量殼聚糖冰乙酸溶液(CHS-L)、1%的中分子量殼聚糖(MW190,000～310,000)冰乙酸溶液(CHS-M)及0.1%的膠原(MW300,000)冰乙酸溶液(COL)分別透析至溶液pH呈中性。分別將CHS-L和CHS-M與COL以2：1和4：1的體積比混合,得到四種混合液�；旌弦航�(jīng)4℃靜置過夜除氣泡、-20℃冷凍48h和-80℃預(yù)凍24h后,采用凍干法制備殼聚糖-膠原膜。將膜裁剪至所需大小后,獨立包裝EO滅菌。 (3)將滅菌膜片浸入配好的細胞培養(yǎng)液中,膜表面積與培養(yǎng)液比例為6cm2/ml,用膜浸提液和常規(guī)細胞培養(yǎng)液培養(yǎng)MC3T3-E1細胞,采用MTT實驗評價膜對細胞增殖活性的影響。 (4)將滅菌膜片植入8只新西蘭兔的背部皮下組織內(nèi),手術(shù)后1周、2周、4周、8周各隨機處死2只動物,取出剩余膜材料。通過計算膜植入前后的質(zhì)量差測定膜在動物體內(nèi)的降解率。 結(jié)果：(1)低分子量殼聚糖冰乙酸溶液與膠原水溶液混合制備的A(2：1)、B(1：1)、C(1：2)三組凍干膜,以2：1和1：1比例制備的膜成膜效果較好,以1：2比例制備的膜成膜效果較差(不進行物理性能評價)。拉伸實驗結(jié)果表明,以2：1比例制備的膜機械性能優(yōu)于1：1比例制備的膜；壓汞實驗顯示,以2：1和1：1比例制備的膜存在廣泛的孔隙,孔隙率分別為89.41%和83.92%；通過掃描電鏡觀察,2：1和1：1比例制備的兩組凍干膜孔隙均較Bio-Gide膜大。 (2)2%的低分子量殼聚糖冰乙酸溶液、1%的中分子量殼聚糖冰乙酸溶液和0.1%的膠原冰乙酸溶液分別透析至溶液呈中性后,未見絮狀物質(zhì)析出。殼聚糖溶液可與膠原溶液均勻混合,混合液經(jīng)真空凍干可制成膜。兩種中分子量殼聚糖凍干膜的厚度和密度,均大于低分子量殼聚糖制備的凍干膜。 (3)中分子量殼聚糖制備的兩種凍干膜,對小鼠MC3T3-E1細胞增殖無抑制。殼聚糖與膠原體積比為4：1時,其浸提液對MC3T3-El細胞的增殖表現(xiàn)出顯著的促進作用,特別是從第4天開始,OD490值明顯高于對照組(P0.01)；殼聚糖與膠原體積比為2：1時,其浸提液對MC3T3-E1細胞增殖無顯著影響,OD490值僅稍高于對照組(P0.05)。 (4)皮下埋植實驗顯示,兩種中分子量殼聚糖-膠原凍干膜植入動物皮下組織后未見明顯的炎癥反應(yīng)。兩種殼聚糖-膠原凍干膜在動物體內(nèi)可降解吸收。8周時,殼聚糖-膠原凍干膜(2：1和4：1)的降解率分別為43.83%和26.45%,但未降解的膜材料已破碎或溶脹呈漿狀。 結(jié)論：(1)殼聚糖和膠原混合制備的凍干膜有較好的物理性能和生物相容性。(2)中分子量殼聚糖(MW190,000-310,000)和膠原的冰乙酸溶液分別透析至中性后以體積比4：1混合,經(jīng)真空凍干制備的復(fù)合可吸收膜,具有良好的成膜性、生物相容性及生物降解性,具備了應(yīng)用于引導(dǎo)骨再生技術(shù)的基本條件。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R318.08

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