羧甲基壳聚糖温敏凝胶的制备及其生物相容性研究
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摘要
背景
牙周病是人类口腔中两大最常见的疾病之一,在世界范围内均有较高的患病率。牙周病治疗的最终目的是实现牙周组织的再生,形成牙周新附着。多年来,人们一直在寻求有效增加牙周新附着、促进牙周组织再生的治疗方法,开展了包括牙周骨移植、生长因子应用及引导牙周组织再生治疗术在内等多方面的研究,并取得了一定的进展,但均不能实现牙周组织的完全再生。近年来,用于组织工程的水凝胶生物材料的研制发展迅速。许多研究表明,壳聚糖水凝胶是一类生物相容性好、可体内降解的生物材料,作为牙周组织工程细胞支架具有广泛的应用前景。羧甲基壳聚糖(Carboxymethyl Chitosan,CMCS)是壳聚糖(Chitosan)经羧甲基化而得的一种水溶性多糖。与壳聚糖相比,羧甲基壳聚糖的水溶性提高,且无毒,无抗原性,具有可降解性,生物相容性好等优点,在医药和保健食品方面的用途日益广泛。有关壳聚糖温敏水凝胶材料的研究近年来报道很多,但针对羧甲基壳聚糖温敏凝胶的研究较少。
目的
自制羧甲基壳聚糖温敏凝胶同时选择多种影响因素优化配方组成,研究其理化性能和对小鼠肺成纤维细胞L929的毒性作用,并且通过动物实验探讨其组织相容性,为进一步的临床应用提供基础实验依据。
方法
本研究分为两部分
1羧甲基壳聚糖温敏凝胶的制备
称取的一定量的羧甲基壳聚糖(CMCS)加入一定浓度的盐酸溶液45ml中,(23±1)℃溶解2 h后,称取一定量的甘油磷酸钠(GP)溶解于1mL超纯水中,在连续搅拌下,缓慢滴加入羧甲基壳聚糖滤液中,得到清澈透明的配合物溶液,再继续搅拌20min混合均匀,备用。将装有CMCS/GP配合物溶液的试管置于30℃～70℃水浴中,达到初始凝胶化温度(IGT)后,恒温一定时间,得到水凝胶。通过调节盐酸的浓度、羧甲基壳聚糖的取代度、甘油磷酸钠的浓度以及羧甲基壳聚糖的浓度来研究其对羧甲基壳聚糖温敏凝胶初始凝胶化温度的影响,从而优化配方组成,制备出能在体温环境下发生凝胶化的医用材料。
2评价羧甲基壳聚糖温敏凝胶的生物相容性
2.1羧甲基壳聚糖温敏凝胶对小鼠肺成纤维细胞L929的细胞毒性作用采用四甲基偶氮唑盐(MTT)法测定不同浓度的羧甲基壳聚糖温敏凝胶浸提液对体外培养的L929细胞的细胞毒性。测定加样后24h、48h、72h、96h的吸光度值(OD),计算细胞相对增殖率(RGR),评定细胞毒性等级。
2.2小鼠皮下埋植实验常规消毒、铺单,在小鼠腿部外侧皮肤切开长1cm的创口,将0.5ml的羧甲基壳聚糖温敏凝胶植入小鼠皮下,空白对照组小鼠在埋植的相同部位除不植入凝胶外,其余步骤相同。缝合筋膜、皮肤。观察各组小鼠在注射凝胶后植入材料周围肌肉组织的外观,然后分别在3、7、14天处死。切除植入部位及周围组织放入福尔马林液固定,常规组织切片,HE染色观察凝胶周围组织的炎症反应,评价各组凝胶的组织相容性。
结果
1制备出柔软、湿润并具有能随环境温度的变化发生可逆性的水凝胶。通过正交化实验优化配方组成后,当羧甲基壳聚糖取代度为0.3,羧甲基壳聚糖浓度为5.0%,盐酸浓度为0.1 mol/m3,GP浓度6.3%时,IGT为37℃最为接近人的口腔温度。
2羧甲基壳聚糖温敏凝胶的生物相容性
2.1羧甲基壳聚糖温敏凝胶对小鼠肺成纤维细胞的细胞毒性作用。
2.1.1加样24h后,0.4倍和0.08倍浸提液组与其他各组的OD值比较差异有统计学意义,P<0.05;其余各组OD值比较差异无统计学意义,P>0.05。
2.1.2加样48h后,50倍浸提液组与阴性对照组及阳性对照组OD值比较差异无统计学意义,P>0.05;其余各实验组的OD值与阴性对照组比较差异有统计学意义,P<0.05。
2.1.3加样72h后,50倍浸提液组与阴性对照组OD值比较差异无统计学意义,P>0.05;50倍浸提液组及阴性对照组与阳性性对照组OD值比较差异有统计学意义,P<0.05;其余各实验组OD值与阴性对照组比较差异有统计学意义,P<0.05。
2.1.4加样96h后,50倍浸提液组与阴性对照组OD值比较差异有统计学意义,P<0.05;50倍浸提液组及阴性对照组与阳性性对照组OD值比较差异有统计学意义,P<0.05;其余各实验组OD值与阴性对照组比较差异有统计学意义, P<0.05。
2.2小鼠皮下埋植实验
本实验组织学表明,凝胶注射后前3d,实验组以急性炎症为主,中性粒细胞大量聚集,淋巴细胞较多,并见巨噬细胞,毛细血管充血;凝胶注射7d后,实验组有少量波浪形胶原纤维形成,毛细血管减少,血管充血程度降低,炎症细胞的数量明显减少,未发现新生的毛细血管;到14d实验组炎症细胞基本消失。说明羧甲基温敏凝胶具有很好的组织相容性。
结论
1研究羧甲基壳聚糖温敏凝胶的制备方法,制备出在室温下为液体,在体温环境下发生凝胶化的具有温敏特性的医用材料。
2对羧甲基壳聚糖温敏凝胶的生物相容性进行了初步评价,证明其对L929成纤维细胞没有显示出毒性,并且有促进细胞增殖的作用,其中0.4倍浸提液促细胞增殖作用最为明显。小鼠皮下埋植实验结果表明14d后即看不到炎症细胞并与正常对照组无明显差别,说明羧甲基壳聚糖温敏凝胶具有良好的组织相容性。
Background
Periodontal disease is one of the most common diseases in human oral cavity, and has comparatively high prevalence in the world. The final purpose of curing periodontal disease is to fulfill the periodontal regeneration and form periodontal new attachment. For many years, people are seeking the method to increase periodontal new attachment effectively and accelerate the periodontal regeneration. In order to get this goales people has made periodontal bone transplantation, growth factor application and guide periodontal tissue regeneration curing method. Although the researches have achieved certain development, these can still hardly fulfill the complete periodontal tissue regeneration. In recent years research on tissue engineer using hydrogel biomaterials has developed rapidly. It is proved by many researches that chitosan hydrogel is a kind of biomaterial with good biocompatibility and degradability in vivo and bears universal application prospect as the support of periodontal tissue engineer cell. Carboxymethyl Chitosan (CMCS) is water-soluble polysaccharide turned from chitosan by carboxy methylation. Compared with chitosan, carboxymethyl chitosan is of higher water-solubility, nontoxic, antigenic free, degradable and good biocompatibility, and it is used widely in medicine and health food. There are many reports about the research on chitosan temperature sensitive gel in recent years, but research focusing on carboxymethyl temperature sensitive gel is rather few.
Objective
Choosing various influencing factor to optimize formula composition while self make carboxymethyl chitosan temperature sensitive gel, and studying its physicochemical property and its toxicity on mice’s lung fibroblasts L929, moreover, probe into its tissue compatibility through animal experiments which can provide the fundamental experiment proof for further clinical application.
Methods
The research can be divided into two parts
1 Preparation of carboxymethyl chitosan temperature sensitive gel The CMCS disinfected withγRay and weigh it certain amount, then put it into 45ml hydrochloride solution, and dissolute for 2h in temperature(23±1)℃. Weigh certain amount of GP to dissolute it into ultra pure water, and add them slowly into Carboxymethyl Chitosan filtrate with continuous mixing, then the clear and transparent complex solution comes into being. Continue mix it even for 20mix for back up. Take the test tube with CMCS/GP complex solution into water bath with temperature 30℃～70℃until it reaches the initial gelation temperature(IGT), then keep the same temperature for a while to get hydrogel. Through adjust hydrochloric acid concentration, substitution degree of carboxymethyl, GP concentration and carboxymethyl concentration research on their effect toward carboxymethyl chitosan temperature sensitive gelcan be achieved, so as to optimize the formula composition and make the medical material which can be gelatificated under the body temperature.
2 Evaluate the biocompatibility of carboxymethyl chitosan temperature sensitive gel
2.1 Effect of cell toxicity of carboxymethyl chitosan temperature sensitive gel on mice lung fibroblasts L929
Adopt MTT method to measure the cell toxicity that carboxymethyl chitosan temperature sensitive gels of different concentration produced on L929 cell cultured in vitro. Measure the OD value after 24h, 48h, 72h, and 96h and calculate RGR, then calculate the cell toxicity grade.
2.2 The subcutaneous implantation of the mouse
Carrying out regular disinfection, paving the table, and cuting a wound with 1cm long on the outside skin of mice’s leg, and transplant carboxymethyl chitosan temperature sensitive gel 0.5ml in its subcutaneous tissue, while no transplantation is carried out on the blank comparison mice. Then suture fascia and skin. Observe the appearance difference of each experiment mice group on its muscle tissue surrounding where gel were injected, and kill them separately on the third, 7th and 14th day. Remove the transplantation part together with the surrounding tissue, and put them into formalin to be fixed, and make regular tissue slice, then HE dye and observe the inflammatory response on the surrounding tissue of gel, finally evaluate the tissue biocompatibility and degradability of the gel of each group.
Results
1 The hydrogel which we prepared has many characteristics, it is a soft, moist and with the ambient temperature changes can occur reversibly. After formula composition is optimized by the orthogonal experiment. When the degree of substitution(DS) of CMCS is 0.3, the concentration of CMCS is 5.0%, the concentration of HCL is 0.1 mol/m3, the concentration of GP is 6.3%, IGT is 37℃which is the closest person oral temperature.
2 The biocompatibility of the Carboxymethyl chitosan thermosensitive hydrogel.
2.1 To evaluate the the cytotoxicity effects of the Carboxymethyl chitosan thermosensitive hydrogel on mouse lung fibroblasts(L929).
2.1.1 After dosing 24h, it is statistical difference between 0.4 times group and other groups, 0.08 times group and other groups, P<0.05; it is no statistical difference between the other experimental groups, P>0.05.
2.1.2 After dosing 48h, it is no statistical difference between 50 times group and negative group, 50 times group and positive group, P>0.05; it is statistical difference between the other experimental groups and negative group, P<0.05.
2.1.3 After dosing 72h, it is no statistical difference between 50 times group and negative group, P>0.05; it is statistical difference between 50 times group and positive group, negative group and positive group. P<0.05; it is statistical difference between the other experimental groups and negative group, P<0.05.
2.1.4 After dosing 96h, it is statistical difference between 50 times group and negative group, P<0.05; it is statistical difference between 50 times group and positive group, negative group and positive group, P<0.05; it is statistical difference between the other experimental groups and negative group, P<0.05.
2.2 The subcutaneous implantation of the mouse
The research histology shows that, on the 3dth after histology injecting, the experimental group is mainly in the acute inflammation, the Large accumulation of neutrophils, lymphocytes is more, and see macrophages and capillary congestion; on the 7dth after histology injecting, the experimental group had a small amount of wavy collagen fibers, capillaries decreased, vascular congestion is decreased, the number of inflammatory cells are significantly reduced, newborn capillaries are not found; on the 14dth after histology injecting, The Inflammatory cells disappeared in the experimental group, it descriptions that carboxymethyl thermosensitive hydrogels have good biocompatibility.
Conclusion
1 Studying on preparation technique of carboxymethyl chitosan, it was prepared by a substance that is liquid at room temperature, it will become gelatination under the body temperature environment and it have thermosensitive characteristic medical material.
2 We have carried on the preliminary appraisal for biocompatibility of Carboxymethyl chitosan thermosensitive hydrogel, and proved that it has no toxicity to L929 cells, in which 0.4 times leaching liquor have the cell-multiplication function obviously. After the experiment of Hypodermic buries plants to mouse for 14 days, we can not see the obvious difference between inflammatory cells with compare group, which can proved Carboxymethyl chitosan thermosensitive hydrogel have well biocompatibility.
牙周病是人类口腔中两大最常见的疾病之一,在世界范围内均有较高的患病率。牙周病治疗的最终目的是实现牙周组织的再生,形成牙周新附着。多年来,人们一直在寻求有效增加牙周新附着、促进牙周组织再生的治疗方法,开展了包括牙周骨移植、生长因子应用及引导牙周组织再生治疗术在内等多方面的研究,并取得了一定的进展,但均不能实现牙周组织的完全再生。近年来,用于组织工程的水凝胶生物材料的研制发展迅速。许多研究表明,壳聚糖水凝胶是一类生物相容性好、可体内降解的生物材料,作为牙周组织工程细胞支架具有广泛的应用前景。羧甲基壳聚糖(Carboxymethyl Chitosan,CMCS)是壳聚糖(Chitosan)经羧甲基化而得的一种水溶性多糖。与壳聚糖相比,羧甲基壳聚糖的水溶性提高,且无毒,无抗原性,具有可降解性,生物相容性好等优点,在医药和保健食品方面的用途日益广泛。有关壳聚糖温敏水凝胶材料的研究近年来报道很多,但针对羧甲基壳聚糖温敏凝胶的研究较少。
目的
自制羧甲基壳聚糖温敏凝胶同时选择多种影响因素优化配方组成,研究其理化性能和对小鼠肺成纤维细胞L929的毒性作用,并且通过动物实验探讨其组织相容性,为进一步的临床应用提供基础实验依据。
方法
本研究分为两部分
1羧甲基壳聚糖温敏凝胶的制备
称取的一定量的羧甲基壳聚糖(CMCS)加入一定浓度的盐酸溶液45ml中,(23±1)℃溶解2 h后,称取一定量的甘油磷酸钠(GP)溶解于1mL超纯水中,在连续搅拌下,缓慢滴加入羧甲基壳聚糖滤液中,得到清澈透明的配合物溶液,再继续搅拌20min混合均匀,备用。将装有CMCS/GP配合物溶液的试管置于30℃～70℃水浴中,达到初始凝胶化温度(IGT)后,恒温一定时间,得到水凝胶。通过调节盐酸的浓度、羧甲基壳聚糖的取代度、甘油磷酸钠的浓度以及羧甲基壳聚糖的浓度来研究其对羧甲基壳聚糖温敏凝胶初始凝胶化温度的影响,从而优化配方组成,制备出能在体温环境下发生凝胶化的医用材料。
2评价羧甲基壳聚糖温敏凝胶的生物相容性
2.1羧甲基壳聚糖温敏凝胶对小鼠肺成纤维细胞L929的细胞毒性作用采用四甲基偶氮唑盐(MTT)法测定不同浓度的羧甲基壳聚糖温敏凝胶浸提液对体外培养的L929细胞的细胞毒性。测定加样后24h、48h、72h、96h的吸光度值(OD),计算细胞相对增殖率(RGR),评定细胞毒性等级。
2.2小鼠皮下埋植实验常规消毒、铺单,在小鼠腿部外侧皮肤切开长1cm的创口,将0.5ml的羧甲基壳聚糖温敏凝胶植入小鼠皮下,空白对照组小鼠在埋植的相同部位除不植入凝胶外,其余步骤相同。缝合筋膜、皮肤。观察各组小鼠在注射凝胶后植入材料周围肌肉组织的外观,然后分别在3、7、14天处死。切除植入部位及周围组织放入福尔马林液固定,常规组织切片,HE染色观察凝胶周围组织的炎症反应,评价各组凝胶的组织相容性。
结果
1制备出柔软、湿润并具有能随环境温度的变化发生可逆性的水凝胶。通过正交化实验优化配方组成后,当羧甲基壳聚糖取代度为0.3,羧甲基壳聚糖浓度为5.0%,盐酸浓度为0.1 mol/m3,GP浓度6.3%时,IGT为37℃最为接近人的口腔温度。
2羧甲基壳聚糖温敏凝胶的生物相容性
2.1羧甲基壳聚糖温敏凝胶对小鼠肺成纤维细胞的细胞毒性作用。
2.1.1加样24h后,0.4倍和0.08倍浸提液组与其他各组的OD值比较差异有统计学意义,P<0.05;其余各组OD值比较差异无统计学意义,P>0.05。
2.1.2加样48h后,50倍浸提液组与阴性对照组及阳性对照组OD值比较差异无统计学意义,P>0.05;其余各实验组的OD值与阴性对照组比较差异有统计学意义,P<0.05。
2.1.3加样72h后,50倍浸提液组与阴性对照组OD值比较差异无统计学意义,P>0.05;50倍浸提液组及阴性对照组与阳性性对照组OD值比较差异有统计学意义,P<0.05;其余各实验组OD值与阴性对照组比较差异有统计学意义,P<0.05。
2.1.4加样96h后,50倍浸提液组与阴性对照组OD值比较差异有统计学意义,P<0.05;50倍浸提液组及阴性对照组与阳性性对照组OD值比较差异有统计学意义,P<0.05;其余各实验组OD值与阴性对照组比较差异有统计学意义, P<0.05。
2.2小鼠皮下埋植实验
本实验组织学表明,凝胶注射后前3d,实验组以急性炎症为主,中性粒细胞大量聚集,淋巴细胞较多,并见巨噬细胞,毛细血管充血;凝胶注射7d后,实验组有少量波浪形胶原纤维形成,毛细血管减少,血管充血程度降低,炎症细胞的数量明显减少,未发现新生的毛细血管;到14d实验组炎症细胞基本消失。说明羧甲基温敏凝胶具有很好的组织相容性。
结论
1研究羧甲基壳聚糖温敏凝胶的制备方法,制备出在室温下为液体,在体温环境下发生凝胶化的具有温敏特性的医用材料。
2对羧甲基壳聚糖温敏凝胶的生物相容性进行了初步评价,证明其对L929成纤维细胞没有显示出毒性,并且有促进细胞增殖的作用,其中0.4倍浸提液促细胞增殖作用最为明显。小鼠皮下埋植实验结果表明14d后即看不到炎症细胞并与正常对照组无明显差别,说明羧甲基壳聚糖温敏凝胶具有良好的组织相容性。
Background
Periodontal disease is one of the most common diseases in human oral cavity, and has comparatively high prevalence in the world. The final purpose of curing periodontal disease is to fulfill the periodontal regeneration and form periodontal new attachment. For many years, people are seeking the method to increase periodontal new attachment effectively and accelerate the periodontal regeneration. In order to get this goales people has made periodontal bone transplantation, growth factor application and guide periodontal tissue regeneration curing method. Although the researches have achieved certain development, these can still hardly fulfill the complete periodontal tissue regeneration. In recent years research on tissue engineer using hydrogel biomaterials has developed rapidly. It is proved by many researches that chitosan hydrogel is a kind of biomaterial with good biocompatibility and degradability in vivo and bears universal application prospect as the support of periodontal tissue engineer cell. Carboxymethyl Chitosan (CMCS) is water-soluble polysaccharide turned from chitosan by carboxy methylation. Compared with chitosan, carboxymethyl chitosan is of higher water-solubility, nontoxic, antigenic free, degradable and good biocompatibility, and it is used widely in medicine and health food. There are many reports about the research on chitosan temperature sensitive gel in recent years, but research focusing on carboxymethyl temperature sensitive gel is rather few.
Objective
Choosing various influencing factor to optimize formula composition while self make carboxymethyl chitosan temperature sensitive gel, and studying its physicochemical property and its toxicity on mice’s lung fibroblasts L929, moreover, probe into its tissue compatibility through animal experiments which can provide the fundamental experiment proof for further clinical application.
Methods
The research can be divided into two parts
1 Preparation of carboxymethyl chitosan temperature sensitive gel The CMCS disinfected withγRay and weigh it certain amount, then put it into 45ml hydrochloride solution, and dissolute for 2h in temperature(23±1)℃. Weigh certain amount of GP to dissolute it into ultra pure water, and add them slowly into Carboxymethyl Chitosan filtrate with continuous mixing, then the clear and transparent complex solution comes into being. Continue mix it even for 20mix for back up. Take the test tube with CMCS/GP complex solution into water bath with temperature 30℃～70℃until it reaches the initial gelation temperature(IGT), then keep the same temperature for a while to get hydrogel. Through adjust hydrochloric acid concentration, substitution degree of carboxymethyl, GP concentration and carboxymethyl concentration research on their effect toward carboxymethyl chitosan temperature sensitive gelcan be achieved, so as to optimize the formula composition and make the medical material which can be gelatificated under the body temperature.
2 Evaluate the biocompatibility of carboxymethyl chitosan temperature sensitive gel
2.1 Effect of cell toxicity of carboxymethyl chitosan temperature sensitive gel on mice lung fibroblasts L929
Adopt MTT method to measure the cell toxicity that carboxymethyl chitosan temperature sensitive gels of different concentration produced on L929 cell cultured in vitro. Measure the OD value after 24h, 48h, 72h, and 96h and calculate RGR, then calculate the cell toxicity grade.
2.2 The subcutaneous implantation of the mouse
Carrying out regular disinfection, paving the table, and cuting a wound with 1cm long on the outside skin of mice’s leg, and transplant carboxymethyl chitosan temperature sensitive gel 0.5ml in its subcutaneous tissue, while no transplantation is carried out on the blank comparison mice. Then suture fascia and skin. Observe the appearance difference of each experiment mice group on its muscle tissue surrounding where gel were injected, and kill them separately on the third, 7th and 14th day. Remove the transplantation part together with the surrounding tissue, and put them into formalin to be fixed, and make regular tissue slice, then HE dye and observe the inflammatory response on the surrounding tissue of gel, finally evaluate the tissue biocompatibility and degradability of the gel of each group.
Results
1 The hydrogel which we prepared has many characteristics, it is a soft, moist and with the ambient temperature changes can occur reversibly. After formula composition is optimized by the orthogonal experiment. When the degree of substitution(DS) of CMCS is 0.3, the concentration of CMCS is 5.0%, the concentration of HCL is 0.1 mol/m3, the concentration of GP is 6.3%, IGT is 37℃which is the closest person oral temperature.
2 The biocompatibility of the Carboxymethyl chitosan thermosensitive hydrogel.
2.1 To evaluate the the cytotoxicity effects of the Carboxymethyl chitosan thermosensitive hydrogel on mouse lung fibroblasts(L929).
2.1.1 After dosing 24h, it is statistical difference between 0.4 times group and other groups, 0.08 times group and other groups, P<0.05; it is no statistical difference between the other experimental groups, P>0.05.
2.1.2 After dosing 48h, it is no statistical difference between 50 times group and negative group, 50 times group and positive group, P>0.05; it is statistical difference between the other experimental groups and negative group, P<0.05.
2.1.3 After dosing 72h, it is no statistical difference between 50 times group and negative group, P>0.05; it is statistical difference between 50 times group and positive group, negative group and positive group. P<0.05; it is statistical difference between the other experimental groups and negative group, P<0.05.
2.1.4 After dosing 96h, it is statistical difference between 50 times group and negative group, P<0.05; it is statistical difference between 50 times group and positive group, negative group and positive group, P<0.05; it is statistical difference between the other experimental groups and negative group, P<0.05.
2.2 The subcutaneous implantation of the mouse
The research histology shows that, on the 3dth after histology injecting, the experimental group is mainly in the acute inflammation, the Large accumulation of neutrophils, lymphocytes is more, and see macrophages and capillary congestion; on the 7dth after histology injecting, the experimental group had a small amount of wavy collagen fibers, capillaries decreased, vascular congestion is decreased, the number of inflammatory cells are significantly reduced, newborn capillaries are not found; on the 14dth after histology injecting, The Inflammatory cells disappeared in the experimental group, it descriptions that carboxymethyl thermosensitive hydrogels have good biocompatibility.
Conclusion
1 Studying on preparation technique of carboxymethyl chitosan, it was prepared by a substance that is liquid at room temperature, it will become gelatination under the body temperature environment and it have thermosensitive characteristic medical material.
2 We have carried on the preliminary appraisal for biocompatibility of Carboxymethyl chitosan thermosensitive hydrogel, and proved that it has no toxicity to L929 cells, in which 0.4 times leaching liquor have the cell-multiplication function obviously. After the experiment of Hypodermic buries plants to mouse for 14 days, we can not see the obvious difference between inflammatory cells with compare group, which can proved Carboxymethyl chitosan thermosensitive hydrogel have well biocompatibility.
引文
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