五倍子牙周缓释凝胶的研制及体外实验的研究
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摘要
牙周病是多因素疾病,到目前为止,尚无理想的治疗方法。牙菌斑生物膜及其产物是牙周病的始动因子,但细菌的破坏是有限的,由细菌引发的局部及全身的一系列宿主免疫反应才是造成牙周组织破坏的主要原因。而牙周疾病所造成的牙周附着丧失一直是临床治疗中面临的一个非常棘手的难题。
药物治疗作为牙周治疗中一种不可或缺的手段,常常用于机械刮治术后以提高和巩固疗效。其中局部缓释剂能直接作用于病变组织,长时间保持局部有效药物浓度等诸多优点而成为牙周病治疗的主要用药方式,但目前临床使用的牙周缓释剂还存在诸多不足,尚无一种可达到理想标准,难以满足临床要求。
天然药物是近年来研究的热点,其中五倍子具有抗菌,止血,收敛等多种功效。已有研究表明,五倍子对牙周可疑致病菌有抑制作用;能拮抗LPS对牙周膜细胞的损伤,并促进其增殖和在牙骨质片上的附着;能通过抑制胶原酶的活性阻止胶原的降解。聚乳酸-羟基乙酸共聚物(PLGA)是可生物降解的高分子缓释载体,随着材料在体内的逐渐降解,复合在载体材料中的药物则缓慢释放,达到持续作用的目的。
本研究拟将五倍子水提取物与PLGA相结合,研制一种理化性质符合临床需要的缓释凝胶制剂,并通过体外培养HPDLFs,考察该制剂对细胞增殖,ALP活性及MMP-3表达的影响,初步探讨其用于牙周病治疗的可行性。本研究分四个实验:
实验一五倍子牙周缓释凝胶配比的初步测定和药物释放度检测
将不同浓度的PLGA和不同的溶剂配比制成五倍子缓释凝胶,观察其溶解、凝结时间及理化性状,并进行体外释放度测定,考察各因素对凝胶中药物体外释放的影响。结果显示:混合溶剂中1MP的含量越多,PLGA溶解的越快,GTA的含量越多,制备出的凝胶流动性越小,凝胶遇水凝结时间就越长;而在相同比例的混合溶剂中,PLGA浓度越高,制成的凝胶越黏稠,遇水凝结时间也越短。凝胶体外释放度测定结果表明药物的释放速度与混合溶剂中1MP比例成正比,与GTA的比例及PLGA的浓度成反比,同时药物浓度越高,初释率也越高;所有制剂7d的累计释放率都超过了80%。综合各项因素,我们初步确定1MP∶GTA = 7∶3,PLGA浓度为40%,五倍子浓度为8%为缓释凝胶配比的参考值。
实验二五倍子牙周缓释凝胶的配方筛选及质量考察
本实验将PLGA浓度、溶剂配比和五倍子浓度按三因素三水平作正交设计,进行体外释放度试验,以第6d的释放百分率为指标,计算极差,以探寻五倍子牙周缓释凝胶的最优配方,同时进行凝胶的质量考察。结果显示:按极差大小来判断,三个因素对凝胶影响大小的顺序为PLGA浓度>溶剂配比>五倍子浓度,最佳配方为PLGA浓度35%,1MP∶GTA = 7∶3,五倍子浓度为4%。经过质量考察说明该凝胶质地均匀,细腻,有一定粘稠度且稳定性好。
实验三五倍子牙周缓释凝胶对LPS抑制HPDLFs增殖及ALP活性的影响
采用细胞培养技术,MTT法,酶联免疫技术观察五倍子牙周缓释凝胶对LPS抑制HPDLFs增殖及ALP活性的影响。结果显示:100μg/mL LPS可显著抑制HPDLFs的增殖和ALP活性。而加入五倍子凝胶释放液后,与LPS组相比,各时间点凝胶释放液组的细胞增殖活性和ALP活性均有所提高,其中第3d释放液组的效果最明显。此结果说明五倍子牙周缓释凝胶可有效拮抗LPS对细胞的抑制作用。
实验四五倍子牙周缓释凝胶对LPS介导HPDLFs分泌MMP-3的影响
本实验体外培养细胞,运用ELISA分别检测单纯LPS作用组和LPS加不同时间点凝胶释放液组下HPDLFs分泌MMP-3的量。结果显示:与空白对照组相比,50μg/m L的LPS即能明显促进HPDLFs分泌MMP-3(P<0.05),100μg/m L的LPS促进作用更显著;加入五倍子凝胶释放液后,各时间点凝胶释放液组MMP-3的量均明显低于LPS组(P<0.05),而且,各时间点凝胶释放液组之间亦有显著性差异(P<0.05),其中,2h释放液组MMP-3最低,相对于LPS组,对MMP-3的抑制率为75.77%,随着释放时间的延长,抑制作用逐渐减弱,但第7d释放液仍有32.26%的抑制率。提示:五倍子牙周缓释凝胶可有效抑制LPS介导HPDLFs分泌MMP-3,抑制作用可持续7d。
Periodontitis is a multi-factor disease.There is no ideal treatment to cure it at present. Dental plaque is the initiation factor of periodontitis, but the destruction caused by bacteria is limited. A series of local and systemic reactions triggered by bacteria is the main reason for the destruction of periodontal tissues. The loss of periodontal attachment caused by periodontitis has been a very difficult problem that we have to face in clinical treatment.
Medicine treatmeat, as an indispensable means of periodontal treatments, is often used to enhance and consolidate the effect after scaling. The periodontal local release agent can act directly on lesions, and maintain effective drug concentration for a long time. As a result, it has become the main agent in periodontal treatments. However, sustained-release agents used in clinic still have many deficiencies. No one has reached the ideal standard. So it is difficult to meet the clinical requirements.
Natural medicine has become the focus of researchs in this realm. Galla chinensis has antibacterial, hemostatic, and other convergence effect. Research has shown that it can effectively inhibit the activity of various suspected periodontal bacteria, avoid HPDLFs from the destruction caused by LPS, promote the generation of HPDLFs and its attachment on cementum. It also can prevent the degradation of collagen through the inhibition of collagenase activity. Poly lactic and glycollic acid (PLGA) is a biodegradable polymer controlled release carrier. The efficient drug which is mixed in the material will release slowly with the gradual degradation of PLGA in the body, so it can achieve the purpose of continuous action.
In this study, we will use Galla chinensis combined with PLGA to develop a periodontal sustained-release gel that its physical and chemical properties can be consistent with the clinical needs. The feasibility of the gel using in periodontal treatment will be discussed through the study on its effects on the multiplication, the activities of ALP and the expression of MMP-3 in HPDLFs. This study comprised four tests:
1. The preliminary determination of the matrix ratio of the periodontal sustained-released gel of Galla chinensis and in vitro release study
Different concentrations of PLGA and different ratio of solvent were made into periodontal sustained-released gel of Galla chinensis, and its dissolution, setting time and the physical and chemical properties were observed.At the same time,we measured in vitro release study of various factors on the gel in vitro release of drugs. The results showed that: a mixed solvent 1MP more, PLGA dissolved sooner. The more the content of GTA, prepared by the gel mobility of the poorer, gel condensation water longer .And in the same proportion of mixed solvents, the PLGA density is higher ,the gel which makes more mounts thickly, meets the water set time to be also shorter.Gel in vitro release test results showed that the drug release ratio and the ratio of mixed solvents is proportional to 1MP, and is inversely proportional to the proportion of GTA and the density of PLGA.Simultaneously the higer the drug concentration , the higher rates of early release. All agents accumulated 7d release rate exceeded 80%. Integrated the various factors, we tentatively choose 1MP: GTA = 7:3, PLGA concentration of 40%, 8% Galla chinensis concentration of slow-release gel as a ratio of the reference value.
2. Screening of the periodontal sustained-released gel formulation of Galla chinensis and quality inspection
We made an orthogonal design at 3 levels of 3 factors (concentration of PLGA, portion of solvent and concentration of Galla chinensis) to measure the concentration of Galla chinensis in vitro, and used the releasing rate on the sixth day as a parameter. By calculating the range in order to explore a best formula of periodontal sustained-released gel of Galla chinensis, and inspect the quality of gel.The results indicated that: according to the range, the order of three factors' influence to the gel is PLGA concentration > solvent portion > Galla chinensis concentration. The best formula is : the concentration of PLGA = 35% (w /w) , 1MP:GTA = 7:3 and Galla chinensis’s concentration = 4 % (w /w).The quality of study results showed the homogeneous gel, fine , have a certain viscosity and good stability.
3. Effects of a periodontal sustained-released gel of Galla chinensis on the multiplication and the activities of ALP in HPDLFs inhibited by LPS
We used the cell culture technique, MTT and enzyme kinetics methods to investigate the effects of a periodontal sustained-released gel of Galla chinensis on the multiplication and the activities of ALP of human periodontal ligament fibroblasts(HPDLFs) induced by lipopolysaccharide(LPS).The results indicated that at the concentration of 100μg/mL,LPS could inhibit the multiplication of HPDLFs and the activities of ALP.Compared with the LPS group, the multiplication of HPDLFs and the activities of ALP of all the groups increased after treated with Galla chinensis solutions released from the gel. The most obvious effect could be observed among all the groups.The results suggest that the periodontal sustained- released gel of Galla chinensis can be an effective antagonist of the inhibitory effect of LPS on the HPDLFs.
4. Effects of a periodontal sustained-released gel of Galla chinensis on the secretion of MMP-3 in human periodontal ligament fibroblasts induced by lipopolysaccharide
In this experiment HPDLFs were cultured in vitro and treated with LPS or with LPS + Galla chinensis solutions released from the gel at different time points. The amounts of MMP-3 secreted by HPDLFs were detected by ELISA. The results indicated that significant increases (P <0.05) in MMP-3 level were observed in the groups treated by LPS of the concentration from 50 to 100μg/ml when they were compared with blank group. After treated with Galla chinensis solutions released from the gel, the amounts of MMP-3 of all the groups were significantly lower than that of the LPS group (P<0.05). Significant differences were found among all the groups, the amount of MMP-3 secretion of HPDLFs treated with the solution released from the gel in the first 2 hours was the lowest. Compared with the LPS group, the inhibition ratio of MMP-3 of this solution was 75.77%. But with the extending of releasing time, the inhibitory action was weakening gradually, however, the inhibition ratio still remained as high as 32.26% on the 7th day. These results indicated that the periodontal sustained- released gel of Galla chinensis can significantly inhibit the secretion of MMP-3 in human periodontal ligament fibroblasts induced by LPS, and the inhibitory action can last 7 days.
药物治疗作为牙周治疗中一种不可或缺的手段,常常用于机械刮治术后以提高和巩固疗效。其中局部缓释剂能直接作用于病变组织,长时间保持局部有效药物浓度等诸多优点而成为牙周病治疗的主要用药方式,但目前临床使用的牙周缓释剂还存在诸多不足,尚无一种可达到理想标准,难以满足临床要求。
天然药物是近年来研究的热点,其中五倍子具有抗菌,止血,收敛等多种功效。已有研究表明,五倍子对牙周可疑致病菌有抑制作用;能拮抗LPS对牙周膜细胞的损伤,并促进其增殖和在牙骨质片上的附着;能通过抑制胶原酶的活性阻止胶原的降解。聚乳酸-羟基乙酸共聚物(PLGA)是可生物降解的高分子缓释载体,随着材料在体内的逐渐降解,复合在载体材料中的药物则缓慢释放,达到持续作用的目的。
本研究拟将五倍子水提取物与PLGA相结合,研制一种理化性质符合临床需要的缓释凝胶制剂,并通过体外培养HPDLFs,考察该制剂对细胞增殖,ALP活性及MMP-3表达的影响,初步探讨其用于牙周病治疗的可行性。本研究分四个实验:
实验一五倍子牙周缓释凝胶配比的初步测定和药物释放度检测
将不同浓度的PLGA和不同的溶剂配比制成五倍子缓释凝胶,观察其溶解、凝结时间及理化性状,并进行体外释放度测定,考察各因素对凝胶中药物体外释放的影响。结果显示:混合溶剂中1MP的含量越多,PLGA溶解的越快,GTA的含量越多,制备出的凝胶流动性越小,凝胶遇水凝结时间就越长;而在相同比例的混合溶剂中,PLGA浓度越高,制成的凝胶越黏稠,遇水凝结时间也越短。凝胶体外释放度测定结果表明药物的释放速度与混合溶剂中1MP比例成正比,与GTA的比例及PLGA的浓度成反比,同时药物浓度越高,初释率也越高;所有制剂7d的累计释放率都超过了80%。综合各项因素,我们初步确定1MP∶GTA = 7∶3,PLGA浓度为40%,五倍子浓度为8%为缓释凝胶配比的参考值。
实验二五倍子牙周缓释凝胶的配方筛选及质量考察
本实验将PLGA浓度、溶剂配比和五倍子浓度按三因素三水平作正交设计,进行体外释放度试验,以第6d的释放百分率为指标,计算极差,以探寻五倍子牙周缓释凝胶的最优配方,同时进行凝胶的质量考察。结果显示:按极差大小来判断,三个因素对凝胶影响大小的顺序为PLGA浓度>溶剂配比>五倍子浓度,最佳配方为PLGA浓度35%,1MP∶GTA = 7∶3,五倍子浓度为4%。经过质量考察说明该凝胶质地均匀,细腻,有一定粘稠度且稳定性好。
实验三五倍子牙周缓释凝胶对LPS抑制HPDLFs增殖及ALP活性的影响
采用细胞培养技术,MTT法,酶联免疫技术观察五倍子牙周缓释凝胶对LPS抑制HPDLFs增殖及ALP活性的影响。结果显示:100μg/mL LPS可显著抑制HPDLFs的增殖和ALP活性。而加入五倍子凝胶释放液后,与LPS组相比,各时间点凝胶释放液组的细胞增殖活性和ALP活性均有所提高,其中第3d释放液组的效果最明显。此结果说明五倍子牙周缓释凝胶可有效拮抗LPS对细胞的抑制作用。
实验四五倍子牙周缓释凝胶对LPS介导HPDLFs分泌MMP-3的影响
本实验体外培养细胞,运用ELISA分别检测单纯LPS作用组和LPS加不同时间点凝胶释放液组下HPDLFs分泌MMP-3的量。结果显示:与空白对照组相比,50μg/m L的LPS即能明显促进HPDLFs分泌MMP-3(P<0.05),100μg/m L的LPS促进作用更显著;加入五倍子凝胶释放液后,各时间点凝胶释放液组MMP-3的量均明显低于LPS组(P<0.05),而且,各时间点凝胶释放液组之间亦有显著性差异(P<0.05),其中,2h释放液组MMP-3最低,相对于LPS组,对MMP-3的抑制率为75.77%,随着释放时间的延长,抑制作用逐渐减弱,但第7d释放液仍有32.26%的抑制率。提示:五倍子牙周缓释凝胶可有效抑制LPS介导HPDLFs分泌MMP-3,抑制作用可持续7d。
Periodontitis is a multi-factor disease.There is no ideal treatment to cure it at present. Dental plaque is the initiation factor of periodontitis, but the destruction caused by bacteria is limited. A series of local and systemic reactions triggered by bacteria is the main reason for the destruction of periodontal tissues. The loss of periodontal attachment caused by periodontitis has been a very difficult problem that we have to face in clinical treatment.
Medicine treatmeat, as an indispensable means of periodontal treatments, is often used to enhance and consolidate the effect after scaling. The periodontal local release agent can act directly on lesions, and maintain effective drug concentration for a long time. As a result, it has become the main agent in periodontal treatments. However, sustained-release agents used in clinic still have many deficiencies. No one has reached the ideal standard. So it is difficult to meet the clinical requirements.
Natural medicine has become the focus of researchs in this realm. Galla chinensis has antibacterial, hemostatic, and other convergence effect. Research has shown that it can effectively inhibit the activity of various suspected periodontal bacteria, avoid HPDLFs from the destruction caused by LPS, promote the generation of HPDLFs and its attachment on cementum. It also can prevent the degradation of collagen through the inhibition of collagenase activity. Poly lactic and glycollic acid (PLGA) is a biodegradable polymer controlled release carrier. The efficient drug which is mixed in the material will release slowly with the gradual degradation of PLGA in the body, so it can achieve the purpose of continuous action.
In this study, we will use Galla chinensis combined with PLGA to develop a periodontal sustained-release gel that its physical and chemical properties can be consistent with the clinical needs. The feasibility of the gel using in periodontal treatment will be discussed through the study on its effects on the multiplication, the activities of ALP and the expression of MMP-3 in HPDLFs. This study comprised four tests:
1. The preliminary determination of the matrix ratio of the periodontal sustained-released gel of Galla chinensis and in vitro release study
Different concentrations of PLGA and different ratio of solvent were made into periodontal sustained-released gel of Galla chinensis, and its dissolution, setting time and the physical and chemical properties were observed.At the same time,we measured in vitro release study of various factors on the gel in vitro release of drugs. The results showed that: a mixed solvent 1MP more, PLGA dissolved sooner. The more the content of GTA, prepared by the gel mobility of the poorer, gel condensation water longer .And in the same proportion of mixed solvents, the PLGA density is higher ,the gel which makes more mounts thickly, meets the water set time to be also shorter.Gel in vitro release test results showed that the drug release ratio and the ratio of mixed solvents is proportional to 1MP, and is inversely proportional to the proportion of GTA and the density of PLGA.Simultaneously the higer the drug concentration , the higher rates of early release. All agents accumulated 7d release rate exceeded 80%. Integrated the various factors, we tentatively choose 1MP: GTA = 7:3, PLGA concentration of 40%, 8% Galla chinensis concentration of slow-release gel as a ratio of the reference value.
2. Screening of the periodontal sustained-released gel formulation of Galla chinensis and quality inspection
We made an orthogonal design at 3 levels of 3 factors (concentration of PLGA, portion of solvent and concentration of Galla chinensis) to measure the concentration of Galla chinensis in vitro, and used the releasing rate on the sixth day as a parameter. By calculating the range in order to explore a best formula of periodontal sustained-released gel of Galla chinensis, and inspect the quality of gel.The results indicated that: according to the range, the order of three factors' influence to the gel is PLGA concentration > solvent portion > Galla chinensis concentration. The best formula is : the concentration of PLGA = 35% (w /w) , 1MP:GTA = 7:3 and Galla chinensis’s concentration = 4 % (w /w).The quality of study results showed the homogeneous gel, fine , have a certain viscosity and good stability.
3. Effects of a periodontal sustained-released gel of Galla chinensis on the multiplication and the activities of ALP in HPDLFs inhibited by LPS
We used the cell culture technique, MTT and enzyme kinetics methods to investigate the effects of a periodontal sustained-released gel of Galla chinensis on the multiplication and the activities of ALP of human periodontal ligament fibroblasts(HPDLFs) induced by lipopolysaccharide(LPS).The results indicated that at the concentration of 100μg/mL,LPS could inhibit the multiplication of HPDLFs and the activities of ALP.Compared with the LPS group, the multiplication of HPDLFs and the activities of ALP of all the groups increased after treated with Galla chinensis solutions released from the gel. The most obvious effect could be observed among all the groups.The results suggest that the periodontal sustained- released gel of Galla chinensis can be an effective antagonist of the inhibitory effect of LPS on the HPDLFs.
4. Effects of a periodontal sustained-released gel of Galla chinensis on the secretion of MMP-3 in human periodontal ligament fibroblasts induced by lipopolysaccharide
In this experiment HPDLFs were cultured in vitro and treated with LPS or with LPS + Galla chinensis solutions released from the gel at different time points. The amounts of MMP-3 secreted by HPDLFs were detected by ELISA. The results indicated that significant increases (P <0.05) in MMP-3 level were observed in the groups treated by LPS of the concentration from 50 to 100μg/ml when they were compared with blank group. After treated with Galla chinensis solutions released from the gel, the amounts of MMP-3 of all the groups were significantly lower than that of the LPS group (P<0.05). Significant differences were found among all the groups, the amount of MMP-3 secretion of HPDLFs treated with the solution released from the gel in the first 2 hours was the lowest. Compared with the LPS group, the inhibition ratio of MMP-3 of this solution was 75.77%. But with the extending of releasing time, the inhibitory action was weakening gradually, however, the inhibition ratio still remained as high as 32.26% on the 7th day. These results indicated that the periodontal sustained- released gel of Galla chinensis can significantly inhibit the secretion of MMP-3 in human periodontal ligament fibroblasts induced by LPS, and the inhibitory action can last 7 days.
引文
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