本文關(guān)鍵詞： 海藻酸鈣水凝膠 人牙周膜細(xì)胞 納米銀 生物安全性能 成骨能力　出處：《南方醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的本課題旨在制備有助于口腔牙周骨組織再生的可載藥海藻酸鈣凝膠及其納米復(fù)合材料,檢測(cè)材料物理性能,同時(shí)檢測(cè)體內(nèi)和體外生物學(xué)性能,最后研究復(fù)合材料的抗菌能力。為后期進(jìn)行3D打印復(fù)合支架的研究和其臨床應(yīng)用奠定基礎(chǔ)。材料與方法材料:海藻酸鈉粉末、氯化鈣粉末、牛血清白蛋白、高糖培養(yǎng)基、胎牛血清、青鏈霉素抗菌劑、膠原酶、PBS溶液、二甲基亞砜、MTT檢測(cè)試劑盒、成品納米銀、LB營(yíng)養(yǎng)培養(yǎng)基、蘇木素、伊紅、二甲苯、冰凍切片機(jī)、倒置顯微鏡、Micro-CT 機(jī)。方法:離子交聯(lián)法制備海藻酸鈣水凝膠后進(jìn)行成分檢測(cè);使用BCA比色法測(cè)量材料蛋白釋放量;監(jiān)測(cè)干重與濕重進(jìn)行吸水率測(cè)定;分別測(cè)量3天、7天、14天、28天及56天時(shí)預(yù)定時(shí)間點(diǎn)的干重及濕重,分析材料降解情況;進(jìn)行牙周膜細(xì)胞原代的培養(yǎng),使用免疫組化染色進(jìn)行鑒定;MTT(四唑鹽)法檢測(cè)人牙周膜細(xì)胞(hPDLCs)的增殖及海藻酸鈣水凝膠對(duì)細(xì)胞毒性;RT-PCR檢測(cè)材料對(duì)hPDLCs炎癥相關(guān)因子表達(dá)以及骨髓基質(zhì)干細(xì)胞成骨相關(guān)基因表達(dá)的影響;茜素紅染色法檢測(cè)礦化能力;通過(guò)肉湯稀釋法測(cè)量納米銀顆粒最低抑菌濃度;離子交聯(lián)法制備海藻酸鈣/納米銀復(fù)合水凝膠材料并對(duì)其成分分析;采用抑菌圈法檢測(cè)納米銀復(fù)合海藻酸鈣水凝膠對(duì)金黃色葡萄球菌的抑菌作用;HE染色觀察海藻酸鈣水凝膠對(duì)軟組織初期炎癥反應(yīng);制備下頜骨缺損模型;采用Micro-CT對(duì)海藻酸鈣水凝膠骨修復(fù)能力評(píng)價(jià)。所有實(shí)驗(yàn)均重復(fù)三次。統(tǒng)計(jì)學(xué)分析采用SPSS 19.0統(tǒng)計(jì)軟件對(duì)數(shù)據(jù)(x±S)進(jìn)行單因素方差分析(one-wayANOVA)分析。若方差齊,則用Dunnett和Bonferronili對(duì)數(shù)據(jù)進(jìn)行兩兩比較分析;若方差不齊,則使用Dunnett' s分析,p0.05表示具有統(tǒng)計(jì)學(xué)差異。結(jié)果成功制備可載藥注射型海藻酸鈣水凝膠;吸水率測(cè)定顯示海藻酸鈣水凝膠顯示出良好的吸水性能;降解實(shí)驗(yàn)結(jié)果表明25 mg/mL濃度的海藻酸鈣水凝膠其降解時(shí)間與骨組織再生成時(shí)間更為匹配;三種濃度海藻酸鈣水凝膠均處于1級(jí),可認(rèn)為材料對(duì)于(?)牙周膜細(xì)胞無(wú)毒性,生物安全性能良好。海藻酸鈣水凝膠及PLA支架材料均促進(jìn)牙周膜細(xì)胞炎癥因子的表達(dá);海藻酸鈣水凝膠共培養(yǎng)后的細(xì)胞形成的礦化結(jié)節(jié)比空白組及PLA組明顯增多;海藻酸鈣水凝膠促進(jìn)成骨相關(guān)因子表達(dá);成功制備海藻酸鈣水凝膠載納米銀材料;復(fù)合了納米銀后材料具備抑菌作用;海藻酸鈣水凝膠導(dǎo)致的軟組織炎癥反應(yīng)輕,且與組織結(jié)合良好;海藻酸鈣水凝膠對(duì)骨組織缺損誘導(dǎo)再生效果較PLA材料好。結(jié)論成功構(gòu)建可載藥海藻酸鈣材料,體內(nèi)及體外實(shí)驗(yàn)驗(yàn)證其應(yīng)用于口腔組織中具有良好生物學(xué)性能;具有促進(jìn)細(xì)胞骨向分化的能力;25 mg/mL的藻酸鈣凝膠效果最好;成功制備出的海藻酸鈣水凝膠載納米銀材料具有抑菌性;有助于預(yù)防傷口感染。
[Abstract]:Objective to prepare calcium alginate gel and its nanocomposites which can be used for oral periodontal bone regeneration, and to test the physical properties and biological properties in vivo and in vitro. Finally, the antibacterial ability of composite materials was studied. It laid a foundation for the study of 3D printing composite scaffold and its clinical application. Materials and methods: sodium alginate powder, calcium chloride powder, bovine serum albumin, high sugar medium, Fetal bovine serum, streptomycin antimicrobial agent, collagenase PBS solution, dimethyl sulfoxide MTT assay kit, nano-silver lb nutrient medium, hematoxylin, eosin, xylene, frozen slicer, Methods: the components of calcium alginate hydrogel were prepared by ion crosslinking method, the protein release was measured by BCA colorimetry, the water absorption was measured by dry weight and wet weight. The dry and wet weights were measured at 3 days, 7 days, 14 days, 28 days and 56 days, respectively. The degradation of the materials was analyzed, and the primary culture of periodontal ligament cells was carried out. Immunohistochemical staining was used to detect the proliferation of human periodontal ligament cells (hPDLCs) and the expression of inflammatory factors related to hPDLCs and osteogenic phase of bone marrow stromal stem cells (BMSCs) were detected by reverse transcription-polymerase chain reaction (RT-PCR) of calcium alginate hydrogel. The effect of gene expression; Alizarin red staining method was used to detect mineralization ability, the minimum inhibitory concentration of silver nanoparticles was measured by broth dilution method, calcium alginate / nano-silver composite hydrogel material was prepared by ion crosslinking method and its composition was analyzed. The bacteriostatic effect of nano-silver composite calcium alginate hydrogel on Staphylococcus aureus was detected by bacteriostasis method and HE staining was used to observe the initial inflammatory response of calcium alginate hydrogel to soft tissue. Micro-CT was used to evaluate the ability of calcium alginate hydrogel to repair bone. All the experiments were repeated three times. The data were analyzed by SPSS 19.0 software. The data were analyzed by univariate ANOVA (one-way ANOVA). Dunnett and Bonferronili were used to compare and analyze the data, if the variance was not uniform, Dunnett's analysis was used to show statistical difference. Results the injectable calcium alginate hydrogel was successfully prepared. The results of degradation test showed that the degradation time of calcium alginate hydrogel at 25 mg/mL concentration was better than that of bone tissue regeneration. All three kinds of calcium alginate hydrogels are in grade 1. Calcium alginate hydrogel and PLA scaffold can promote the expression of inflammatory factors in periodontal ligament cells. Calcium alginate hydrogel promoted the expression of osteoblast-related factors and successfully prepared calcium alginate hydrogel loaded with nano-silver. The composite of nano-silver has bacteriostatic effect, calcium alginate hydrogel causes soft tissue inflammation, and it binds well to the tissue. Conclusion the calcium alginate hydrogel has a better effect on bone tissue defect induction and regeneration than PLA. Conclusion calcium alginate can be successfully constructed and its biological properties in oral tissue have been proved by in vivo and in vitro experiments. The calcium alginate gel with the ability of promoting bone differentiation for 25 mg/mL has the best effect. The calcium alginate hydrogel loaded with silver nanoparticles prepared successfully has bacteriostasis and is helpful to prevent wound infection.
【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】：2017
【分類號(hào)】：R783.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 Rui Yao;Gang Xu;Shuang-Shuang Mao;Hua-Yu Yang;Xin-Ting Sang;Wei Sun;Yi-Lei Mao;;Three-dimensional printing: review of application in medicine and hepatic surgery[J];Cancer Biology & Medicine;2016年04期

2 夏商;姜力銘;陳旭;;組織工程技術(shù)應(yīng)用于牙周膜再生研究進(jìn)展[J];中國(guó)實(shí)用口腔科雜志;2016年09期

3 鐘恩意;劉瑞源;高杰;黃文華;;3D生物打印技術(shù)及其在牙周骨缺損修復(fù)中的應(yīng)用[J];中國(guó)醫(yī)學(xué)物理學(xué)雜志;2016年01期

4 黃奕華;凌均h，

本文編號(hào)：1496681