真菌发酵制备生物材料壳聚糖

利用犁头霉属真菌发酵生产壳聚糖。发现培养温度、时间以及 p H值等因素对壳聚糖产量及性质产生明显影响。p H值影响壳聚糖的脱乙酰程度 ,p H值控制在 4 .5可获得 90 %以上的脱乙酰度 ;2 8～2 9℃和 p H值 5.0条件下 ,壳聚糖的收率达到 780 mg/ L ;随培养时间延长 ,产品分子量迅速下降。扫描电镜分析显示 ,发酵法生产的壳聚糖成膜后 ,与酸碱处理甲壳素得到的产品在微观结构上存在显著差别     

甲壳素/壳聚糖止血机理及应用

甲壳素通过蛋白质介导黏附血小板,形成的甲壳素/血小板复合物加速血纤维蛋白单体的聚合并共同形成凝块;另一方面,甲壳素诱导红细胞聚集,刺激血管收缩,最终形成血栓,封合伤口.壳聚糖的止血机理基本类似于甲壳素,但两者对红细胞的聚集程度和对补体的激活程度存在一定差异.甲壳素独特的三次结晶结构(包括α型和β型)赋予甲壳素优良的止血功能;壳聚糖的止血效果则与它的分子量、脱乙酰度、质子化程度和物理形式等有关.本文综述了甲壳素和壳聚糖的止血机理及应用情况.     

不同脱乙酰度对壳聚糖膜与角膜基质细胞相容性的影响

以分子量为30万．脱乙酰度分别为63．3％、73．7％、83％和97％的壳聚糖制备不同的壳聚糖膜，在不同脱乙酰度的壳聚糖膜上培养兔角膜基质细胞．通过观察角膜基质细胞在不同壳聚糖膜上的生长状态、贴附情况、生长曲线以及乳酸脱氢酶的活性，研究壳聚糖分子脱乙酰度对壳聚糖膜与角膜基质细胞生物相容性的影响。实验结果表明壳聚糖脱乙酰度越高。壳聚糖膜对细胞的损伤越小。越有利于细胞在膜上的生长和贴附，反之．低脱乙酰度的壳聚糖膜与角膜细胞的相容性较差。     

蝇蛆壳提取壳聚糖的综合评价

目的 壳聚糖具有良好的水溶性、生物相容性、生物降解性、成膜性及止血、愈创、消炎等作用,在医用生物材料应用方面具有广阔前景.蝇蛆的皮中含有大量壳聚糖成分,是获得较纯壳聚糖的方式之一.对蝇蛆提取的壳聚糖创伤敷料进行综合评价.方法 按照国家统一标准对蝇蛆壳聚糖进行理化分析(相对分子质量、脱乙酰度和重金属含量)、抑菌、皮肤致敏、细胞毒性和溶血实验.结果 蝇蛆壳聚糖的相对分子质量为266 ku,脱乙酰度为75％,重金属含量小于10-5,细胞毒性总评级为1级,具有较轻细胞毒性.致敏和溶血实验结果表明,蝇蛆壳聚糖材料不具有致敏反应,材料的溶血率为0.769％,小于5％,即没有溶血现象.此外,蝇蛆壳聚糖还具有抑菌作用.结论 蝇蛆壳聚糖具有较好的综合特性,可以满足生物医药方面的要求.     

壳聚糖醋酸溶液对凝血作用的研究

研究了不同脱乙酰度和不同分子量壳聚糖醋酸溶液的凝血作用。发现壳聚糖醋酸溶液使抗凝血液中红细胞发生了明显的聚集和变形。通过不同分子量和脱乙酰度壳聚糖的促红细胞聚集实验,证明了低脱乙酰度壳聚糖(60%~70%)使红细胞聚集效果更好,分子量在105~106范围内作用不十分明显。对血液的凝血酶时间(TT)、凝血酶原时间(PT)、活化部分促凝血酶原激酶时间(APTT)和纤维蛋白原浓度(FIB)的测定结果验证了壳聚糖醋酸溶液凝血机理不依赖于血小板和常规“瀑布”凝血机制。     

壳聚糖结构和分子量对凝血作用的研究

本工作研究了不同脱乙酰度和不同分子量壳聚糖、壳聚糖醋酸生理盐水溶液及羧甲基壳聚糖生理盐水溶液的凝血作用。在壳聚糖醋酸生理盐水溶液作用下，红细胞发生聚集和形变，而且低脱乙酰度现象明显，分子量在10^5～10^6范围内影响不十分明显；羧甲基壳聚糖不能使红细胞在低相对离心力下发生沉降，仅有部分叠连现象；而壳聚糖则不能使红细胞发生聚集和形变。血液凝血酶时间(TT)、凝血酶原时间(PT)、活化部分促凝血酶原激酶时间(APTT)和纤维蛋白原浓度(FIB)验证了壳聚糖、壳聚糖醋酸生理盐水溶液以及羧甲基壳聚糖均不激活凝血因子，且脱乙酰度、分子量以及取代基对这些指标也没有明显地影响。动态凝血实验发现低脱乙酰度壳聚糖能够更好地使血液凝固。     

低取代6-O-羧甲基壳聚糖结构及其抗菌和促进皮肤创面愈合的研究

采用波谱等现代仪器分析方法表征6-O-羧甲基壳聚糖结构和基本性能,并以6-O-羧甲基壳聚糖水溶胶膜为基本模型,研究其抗菌性能和临床促进皮肤创面愈合等效果.结果表明:改性壳聚糖为低取代(取代度为9.5%)、高脱乙酰度(脱乙酰度为83.2%)的碳6位取代羧甲基壳聚糖,其热分解起始温度低于180℃.溶胶膜对大肠埃希氏菌、金黄色葡萄球菌以及铜绿假单胞菌作用60min时的杀灭率分别为＞99.99%、98.54%、＞99.99%.溶胶膜使用时一般无需换药,对浅Ⅱ°烧烫伤、深Ⅱ°烧烫伤、手术切口、取皮区、溃疡等多种创面均具有明显的促愈合、止血、抑菌、控制渗出、减少疤痕形成的作用,且未发现全身及局部致敏反应,亦未见创面组织过度增生现象.     

口腔抗菌领域中壳聚糖及其衍生物的作用

背景：口腔中微生物种类繁多,形成一个复杂的微环境,菌群失调会引发一系列口腔疾病。目前,抗菌药物的使用主要是口服或局部使用,但由于抗生素的迅速分解与释放,病原微生物对抗生素的抗药性不断增强,常导致临床疗效不佳。近年来研究发现,壳聚糖及其衍生物具有良好的抗菌活性,同时随着纳米技术的发展,壳聚糖及其衍生物以不同的形式在抗菌领域的研究较为广泛。目的：针对壳聚糖及其衍生物的主要抗菌机制及其以不同形式在口腔抗菌领域的应用作一综述。方法：应用计算机在PubMed、Web of Science和中国知网数据库检索涉及壳聚糖及其衍生物在口腔抗菌领域中的相关研究,中英文检索词分别为“Chitosan,chitosan derivative,Antibacterial activity,Antibacterial Mechanism,Oral”和“壳聚糖、壳聚糖衍生物、抗菌活性、抗菌机制、口腔”,检索时限为各数据建库至2022年4月。结果与结论：(1)壳聚糖又称脱乙酰甲壳素,是甲壳素脱乙酰基后的初级衍生物,是目前发现的惟一的阳离子碱性多糖。壳聚糖及其衍生物作为纳米抗菌剂在口腔抗菌领域得到广泛研究。(2)壳聚...     

低取代6-0-羧甲基壳聚糖结构及其抗菌和促进皮肤创面愈合的研究

采用波谱等现代仪器分析方法表征6-O-羧甲基壳聚糖结构和基本性能,并以6-O-羧甲基壳聚糖水溶胶膜为基本模型,研究其抗菌性能和临床促进皮肤创面愈合等效果.结果表明改性壳聚糖为低取代(取代度为9.5%)、高脱乙酰度(脱乙酰度为83.2%)的碳6位取代羧甲基壳聚糖,其热分解起始温度低于180℃.溶胶膜对大肠埃希氏菌、金黄色葡萄球菌以及铜绿假单胞菌作用60min时的杀灭率分别为＞99.99%、98.54%、＞99.99%.溶胶膜使用时一般无需换药,对浅Ⅱ°烧烫伤、深Ⅱ°烧烫伤、手术切口、取皮区、溃疡等多种创面均具有明显的促愈合、止血、抑菌、控制渗出、减少疤痕形成的作用,且未发现全身及局部致敏反应,亦未见创面组织过度增生现象.     

浅析壳聚糖的止血作用及机理

壳聚糖（chitosan）是从虾／蟹等甲壳类动物的外壳中提取的几丁质（chitin）经脱乙酰基后而成,其化学名称为B-（1,4）．2．乙酰氨基．2．脱氧．D．葡聚糖。自1881年Bracomno发现几丁质以来,人们对其进行了广泛深入的研究及开发利用,尤其是近十几年来,人们对几丁质／壳聚糖的理化特性和生物特性有了更深入的认识。几丁质／壳聚糖已被证实具有无毒性、无刺激性、无免疫源性、无热源反应、不溶血、无致突变效应、可自然降解及良好的组织相容性等特点,故广泛用于制造人工皮肤、可吸收缝合线、防粘连剂和药物载体等。壳聚糖具有良好的生物相容性和生物可降解性,还具有抗菌、消炎、止血、减少创面渗出和促进创伤组织再生、修复、愈合的作用以及易于形成凝胶的性质,赋予它用作止血材料的良好性能。     

In vitro degradation of chitosan by a commercial enzyme preparation: effect of molecular weight and degree of deacetylation

A commercially available almond emulsin beta-glucosidase preparation has been reported to have chitobiose activity, and can hydrolyze chitin substrates due to a chitinase present in the enzyme preparation. This beta-glucosidase preparation was used to investigate hydrolytic activity on five chitosan samples with different molecular weight and degree of deacetylation. The degree of deacetylation and molecular weight of the chitosan samples were determined using a circular dichroism and a viscometric method, respectively. The hydrolytic activity of this beta-glucosidase preparation on chitosan was monitored viscometrically as the most convenient means of screening. Solutions of chitosan in pH 5.0 acetate buffer were prepared using the different viscosity grades of chitosan. The specific viscosity, measured after addition of beta-glucosidase to the above solutions, decreased dramatically over time in comparison to that of the respective control mixture without enzyme. Eadie-Hofstee plots established that hydrolysis of chitosan by this enzyme preparation obeyed Michaelis-Menten kinetics. Apparent Michaelis-Menten parameters and initial degradation rates were calculated and compared to determine the influences of the degree of deacetylation and molecular weight on the hydrolysis. The results show that higher molecular weight and higher degree of deacetylation chitosans possessed a lower affinity for the enzyme and a slower degradation rate. Faster degradation rates, then, are expected with lower molecular weight and low degree of deacetylation chitosans. Hydrolysis of these chitosan samples confirms the existence of a chitinase in the almond emulsin beta-glucosidase preparation, and further studies are warranted.     

The effect of the degree of chitosan deacetylation on the efficiency of gene transfection

Chitosans with various degrees of deacetylation were synthesized by acetylation with acetic anhydride. These chitosans were evaluated for efficacy of nanoparticle formation, DNA binding efficiency, morphology, and in vitro and in vivo gene transfection efficiency. DNA binding efficacy was reduced as degree of deacetylation was decreased, therefore requiring an increased +/-ratio to effect complete DNA complexation. For chitosan with a molecular weight of 390 kDa, the +/-ratio to achieve complete DNA complexation for degrees of deacetylation of 90%, 70% and 62% was 3.3:1, 5.0:1, and 9.0:1, respectively. The size and morphology of these nanoparticle formulations were not significantly different. The decreased degree of deacetylation results in a decrease in overall luciferase expression levels in HEK293, HeLa, and SW756 cells due to particle destabilization in the presence of serum proteins. However, intramuscular luciferase expression levels increased with decreasing deacetylation over the time points tested. Degree of chitosan deacetylation is an important factor in chitosan-DNA nanoparticle formulation as it affects DNA binding, release and gene transfection efficiency in vitro and in vivo.     

Controlling branching structure formation of the salivary gland by the degree of chitosan deacetylation

The salivary gland is characterized by ramified epithelial branches, a specific tissue structure responsible for saliva production and regulation. To regenerate the salivary gland function, it is important to establish the tissue structure. Chitosan is a deacetylated derivative of chitin with wide biomedical applications. Because of its deacetylated nature, chitosan has different properties when prepared with different degrees of deacetylation (DDA). However, the impact of chitosan DDA on the effect of regulating tissue structure formation remains unexplored. In this study, the embryonic murine submandibular gland (SMG) was used as a model to investigate the role of chitosan DDA in regulating tissue structure formation of the salivary gland. When chitin substrates with different DDA were used, the branching numbers of cultured SMG explants changed. Similar effects were observed in the culture with chitosan prepared using different degrees of acetylation. The mRNA expressions of type I and type III collagen were elevated in SMG explants with enhanced branching morphogenesis, as was the protein level. In addition to the amounts of collagen, type I and type III collagen fibers were spatially present in the epithelial–mesenchymal junction of developing branches in the culture with chitosan of a specific range of DDA. The branch-promoting effect of chitosan DDA was abolished when SMG explants were treated with collagenase, both early in the stage of branch initiation and with the establishment of the branching structure. The branch-promoting effect of chitosan DDA disappeared when antisense oligonucleotides were applied to specifically block type III collagen. This study demonstrates for the first time that DDA of chitosan affects tissue structure formation. The different proportions of side-chain components of chitin derivatives regulate structural formation of cultured SMG, indicating that DDA is an important parameter using chitosan as a biomaterial for tissue structure formation of the salivary glands.     

Characterization and evaluation of chitosan matrix for in vitro growth of MCF-7 breast cancer cell lines

Biodegradable polymer scaffolds were prepared from chitosan with varying degree of deacetylation for in vitro culture of human breast cancer MCF-7 cell lines. These polymers were characterized in terms of functional groups by FTIR and swelling properties. Polymers having high degree of deacetylation showed better swelling properties irrespective of the molecular weight. These polymers were biocompatible and non-toxic towards human epithelial MCF-7 cell lines. Attachment kinetics of MCF-7 cell lines on to polymer scaffold was investigated and it was observed that polymer having high degree of deacetylation favored better cell attachment. In CPIII polymer scaffold having 80% degree of deacetylation, a maximum of 1 millions cells per mg pf polymer were adsorbed within 1h. It appears that high swelling and high degree of deacetylation of chitosan helped in better adsorption of cancer cell lines. The cellular morphology of the attached cells on chitosan matrix was similar to that observed with regular plastic culture with the difference that, cells grew as three-dimensional clumps on chitosan matrix. Polymer having high degree of deacetylation not only favored better adsorption but also showed improved cell growth kinetics. Maximum cell concentration of 6.5 x 10(5) cells/ml was achieved in 5 days culture on CPIII polymer scaffold. The glucose consumption and lactate production pattern of the MCF-7 cell lines on chitosan polymer matrix were similar to that observed on cell growth on tissue culture flask. These results indicate that chitosan scaffold having high degree of deacetylation can be used for three-dimensional growth of MCF-7 cancer cell lines. Such in vitro 3D culture of cancer cells can thus be used as a model for the cytotoxic evaluation of anticancer drugs.     

Rheological characteristics of semi-dilute chitosan solutions

The present work is concerned with experimental results on rheological characteristic properties of semi-dilute chitosan solutions in weakly acetic acid. The flow measurements were conducted on solutions of chitosan of various deacetylation degrees DD (62.8%–86.7%) with changing experimental conditions such as concentration (up to 5 g/100 mL), temperature (20°C–41°C) and shear rate applied (up to 10³s^?1). Chitosan solutions behave generally as a typical non-Newtonian shear-thinning fluid with a characteristic n-parameter lower than 1. The rheological n-parameter decreases with decreasing deacetylation degree and increasing solution concentration but it increases with increasing temperature. Because of strong intermolecular hydrogen bonding even at low concentration the chitosan macromolecules have a tendency to entangle and to network formation. The density of the molecular entanglements in the chitosan solution depends on concentration, temperature and shear rate applied. An effect of the deacetylation degree on the Huggins viscosity constant related to polymer solvent interaction is found. The most important results refer to the activation energy of viscous flow found from Arrhenius plots. The dependence of the activation energy on solution concentration, shear rate and DD is discussed.     

Biocompatibility of thermosensitive chitosan-based hydrogels: an in vivo experimental approach to injectable biomaterials

Chitosan, an amino-polysaccharide obtained from the alkaline deacetylation of chitin, presents an interest as a drug vehicle. Indeed, chitosan solutions containing glycerol-2-phosphate (beta-GP) undergo sol-gel transition at a temperature close to 37 degrees C, which make them suitable for the parenteral administration of drugs. However, before using these chitosan derivatives for biomedical applications, it is important to evaluate their biocompatibility, and particularly to test their inflammatory effects. When injected in the hindpaw of the rat, we have shown that: (i) four chitosan/beta-GP solutions tested triggered a non-specific response, with solutions prepared with chitosans of higher deacetylation degrees yielding a lesser inflammatory reaction and (ii) systemic pretreatment of animals with icatibant, apafant and diphenhydramine did not significantly diminish this response; dexamethasone practically abolished it for all solutions and ketanserine only slightly decreased it in one preparation at two different times. In conclusion, it appears that a higher degree of deacetylation of the chitin chain is desirable for superior biocompatibility.     

Effect of chitosan and carboxymethyl chitosan on fibrinogen structure and blood coagulation

Chitosan has numerous biomedical applications such as tissue engineering scaffolds, drug/gene delivery systems, hemostasis materials, antibacterial materials, wound dressing, etc. In any case, chitosan administered in vivo would positively or passively contact or enter blood tissue. In this situation, the interaction of chitosan with blood components is critical to determine the efficacy and safety of the polymer. In this study, the effect of chitosan with different molecular weight and its derivative carboxymethyl chitosan (CMC) on the structure and function of clotting-related proteins was studied. Specifically, the structural and conformational change of fibrinogen, an important clotting protein, was studied by using UV, fluorescence, and circular dichroism spectroscopy, respectively. Further, the impact of chitosan and CMC on the clotting function was evaluated with activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen time (FT), and thromboelastography (TEG) assays. These results showed that, chitosan and fibrinogen can form complex mainly by electrostatic attraction. As a result, the structure and conformation of fibrinogen are altered by chitosan and CMC. Additionally, the presence of chitosan and CMC has little impact on the values of APTT, PT and FT, but causes significant abnormality in the clotting process by changing TEG parameters. These results provide important insight into the molecular basis for the biological response to chitosan and other biopolymers.