纳米羟基磷灰石悬浮液对人牙周膜成纤维细胞增殖及细胞周期影响的研究
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摘要
目的
1、初步探讨纳米羟基磷灰石悬浮液对人牙周膜成纤维细胞的作用,为纳米羟基磷灰石应用于牙周病的治疗提供依据。
2、为本课题组今后研究纳米及介孔材料载药后对相关成纤维细胞的凋亡影响做准备。
方法
1.利用组织块培养法培养、鉴定并传代人牙周膜成纤维细胞,利用第4-6代成纤维细胞进行实验。
2.实验室制备纳米羟基磷灰石,利用羧甲基纤维素钠、六偏磷酸钠作为分散剂,进行对比,观察两种分散剂对纳米羟基磷灰石的分散效果,并进行简单的细胞培养,对比细胞生长情况,选择合适的分散剂。
3.采用分散剂对纳米羟基磷灰石进行分散,制备l%浓度的纳米羟基磷灰石悬浮液,分别稀释至0.5%、0.25%、0.1%、0.05%、0.025%,并设置单独0.1%浓度的纳米羟基磷灰石组、单独0.1%浓度的羧甲基纤维素钠组、0.005%介孔硅组、0.01%氢氧化钙组及空白对照组,对人牙周膜成纤维细胞各培养24h、48h、72h后,用MTT法对各组细胞增殖情况进行测定,评价各组对人牙周膜成纤维细胞增殖的影响。
4.选取0.5%浓度的悬浮液与各对照组继续细胞培养48h,将各组培养后的细胞,采用流式细胞术检测细胞周期分布情况,分析各组对人牙周膜成纤维细胞的细胞周期影响。
结果
1.成功培养出人牙周膜成纤维细胞,经抗波形丝蛋白、抗角蛋白染色确定：抗波形丝蛋白阳性,抗角蛋白阴性。
2.实验室采用化学沉淀法制备出纳米羟基磷灰石,经粒径检测所得纳米羟基磷灰石粒径在20-40 nm符合本次实验所需。通过比较六偏磷酸钠和羧甲基纤维素钠的对纳米羟基磷灰石的分散效果,可以得出六偏磷酸钠能更好地分散纳米羟基磷灰石,但进行细胞培养时,采用六偏磷酸钠分散纳米羟基磷灰石进行培养却导致了大量人牙周膜成纤维细胞的死亡,而羧甲基纤维素钠分散的纳米羟基磷灰石组细胞生长良好。因此选用羧甲基纤维素钠作为分散剂配置纳米羟基磷灰石悬浮液,进行后续实验。
3.MTT法检测不同浓度的分组以及多重对照组的细胞增殖情况,发现0.5%浓度的纳米羟基磷灰石悬浮液组对细胞增殖情况较其他组效果明显。在相同浓度(0.1%)下,纳米羟基磷灰石悬浮液组对细胞的增殖作用优于单独纳米羟基磷灰石组。
4.采用流式细胞术检测结果显示纳米羟基磷灰石悬浮液组可以减少处于G0/G1期的细胞数量,增加处于S期的细胞数量,使人牙周膜成纤维细胞在细胞增殖周期中很快完成G0/G1,期向s的过渡。
结论
1.纳米羟基磷灰石悬浮液对人牙周膜成纤维细胞的增殖有明显的促进作用。在细胞培养允许的渗透浓度内,浓度越高对细胞增殖的促进作用越明显,而且纳米羟基磷灰石对人牙周膜成纤维细胞增殖的促进作用在前24h最为明显。
2.纳米羟基磷灰石悬浮液作用于人牙周膜成纤维细胞,可以缩短细胞处于Go/G1,的时间,使较大量的细胞进入S期,进行有丝分裂,加速了HPDLCs的增殖速度。
3.通过本研究,进一步肯定了nHA运用于牙周软组织重建的可能,为nHA运用于牙周病的治疗提供了参考依据。
Objectives
1、Discussed the effect on periodontal and cell cycle of human ligament cells by hydroxyapatite suspension in the initial,so as to probe the application of nano-hydroxyapatite used in the treatment of disease.
2、In preparation for the future research on apoptosis of associated fibroblasts by the drug which was loaded with nano and mesoporous materials.
Method
1、Using tissue culture method to culture, identificate and passage human periodontal ligament fibroblasts, the 4th-6th fibroblasts were used for the experiment.
2、Prepared hydroxyapatite in the laboratory,used sodium carboxymethyl cellulose, sodium hexametaphosphate as dispersant. Compared the dispersion between the two dispersants,and compared the cell growth by cell culturing, choosed a suitable dispersant for the next researth.
3、Used the better dispersant which was choosed from the two above to disperse nano-hydroxyapatite.Prepared nano-hydroxyapatite suspension in 1%concentration.then diluted the concentration to 0.5%,0.25%,0.1%, 0.05% and 0.025%. Set some controls just as the blank, sigle hydroxyap-atite group in 0.1% concentration,sodium carboxymethyl cellulose in 0.1% concentration, mesoporous materials in 0.005% concentration and calcium hydroxide in 0.01% concentration.Then cultured the human periodontal ligament fibroblasts for 24 hours,48 huors and 72 huors in mdium with the groups above.And measured the proliferation of cells in each group with MTT to evaluate the proliferation effect of human periodontal ligament fibroblasts.
4^ Selected the suspension in 0.5% concentration as a new group,with the control groups together,human periodontal ligament fibroblasts were went on to culture for 48 huors.Then detected cell cycle distribution by flow cytometry to analysis the impacts of each group on culturing.
Result
1、Successfully cultured human periodontal ligament fibroblasts which identified by anti-vimentin and anti-keratin staining:anti-vimentin-positi-ve,and anti-keratin-negative.
2、Prepared nano-hydroxyapatite by chemical precipitation in laboratory, by testing, the diameter of nano-hydroxyapatite was from 40-60nm,which was required to this experiment. By comparing the sodium hexameta-phosphate and sodium carboxymethyl cellulose on the dispersion of nano-hydroxyapatite, sodium hexametaphosphate could be drawn to better in dispersing hydroxyapatite.However,it led to a large number of dead cells when nano-hydroxyapatite suspension which dispersed by sodium hexametaphosphate that was added in medium to culture human periodontal ligament fibroblasts.But the cells grow well in another group in which nano-hydroxyapatite was dispersed by sodium carboxymethyl cellulose. So sodium carboxymethyl cellulose was choosen to went on reseach as the dispersant we wanted to disperse the nano-hydroxyapatite suspension.
3、Cultured cells in different concentrations and multiple group which set for the control,then used MTT to detect proliferation of cells.The effect of proliferation was found significantly higher than other groups, when nano-hydroxyapatite was dispersed with sodium carboxymethyl cellulose in 0.5% concentration. At the same concentration(0.1%), the effect of cell proliferation was better in nano-hydroxyapatite suspension group than single nano-hydroxyapatite group.
4、The result by flow cytometry showed that it would reduce the number of cells in G0/G1 phase,when cell cultured in the group which nano-hydr-oxyapatite was dispersed in sodium carboxymethyl cellulose.At the same time, the number of cells in S phase would increase. It could be infered that human periodontal ligament fibroblasts would transite G0/G1 to S phase with the effect of nano-hydroxyapatite.
Conclusion
1、Dispersed in sodium carboxymethyl cellulose, nano-hydroxyapatite suspension had a good nano activity in its suspension,and it significantly promoted the proliferation of human periodontal ligament fibroblasts. Within the isotonic concentration, the higher the concentration was, the deeper the effection would be on the cell proliferation,and it led an obvious effect at the first 24 hours.
2、Dispersed in sodium carboxymethyl cellulose, nano-hydroxyapatite suspension acting on human periodontal ligament fibroblasts can shorten the G0/G1 phase time in human periodontal ligament fibroblasts' mitosis cycle, and made relatively large number of cells transite to S phase,for this it accelerated proliferation rate of human periodontal ligament fibroblasts
3、Through this study,It further affirmed that nano-hydroxyapatite can be used for reconstruction of periodontal soft tissue,and it provide a reference for nano-hydroxyapatite using in the treatment of periodontal disease.
1、初步探讨纳米羟基磷灰石悬浮液对人牙周膜成纤维细胞的作用,为纳米羟基磷灰石应用于牙周病的治疗提供依据。
2、为本课题组今后研究纳米及介孔材料载药后对相关成纤维细胞的凋亡影响做准备。
方法
1.利用组织块培养法培养、鉴定并传代人牙周膜成纤维细胞,利用第4-6代成纤维细胞进行实验。
2.实验室制备纳米羟基磷灰石,利用羧甲基纤维素钠、六偏磷酸钠作为分散剂,进行对比,观察两种分散剂对纳米羟基磷灰石的分散效果,并进行简单的细胞培养,对比细胞生长情况,选择合适的分散剂。
3.采用分散剂对纳米羟基磷灰石进行分散,制备l%浓度的纳米羟基磷灰石悬浮液,分别稀释至0.5%、0.25%、0.1%、0.05%、0.025%,并设置单独0.1%浓度的纳米羟基磷灰石组、单独0.1%浓度的羧甲基纤维素钠组、0.005%介孔硅组、0.01%氢氧化钙组及空白对照组,对人牙周膜成纤维细胞各培养24h、48h、72h后,用MTT法对各组细胞增殖情况进行测定,评价各组对人牙周膜成纤维细胞增殖的影响。
4.选取0.5%浓度的悬浮液与各对照组继续细胞培养48h,将各组培养后的细胞,采用流式细胞术检测细胞周期分布情况,分析各组对人牙周膜成纤维细胞的细胞周期影响。
结果
1.成功培养出人牙周膜成纤维细胞,经抗波形丝蛋白、抗角蛋白染色确定：抗波形丝蛋白阳性,抗角蛋白阴性。
2.实验室采用化学沉淀法制备出纳米羟基磷灰石,经粒径检测所得纳米羟基磷灰石粒径在20-40 nm符合本次实验所需。通过比较六偏磷酸钠和羧甲基纤维素钠的对纳米羟基磷灰石的分散效果,可以得出六偏磷酸钠能更好地分散纳米羟基磷灰石,但进行细胞培养时,采用六偏磷酸钠分散纳米羟基磷灰石进行培养却导致了大量人牙周膜成纤维细胞的死亡,而羧甲基纤维素钠分散的纳米羟基磷灰石组细胞生长良好。因此选用羧甲基纤维素钠作为分散剂配置纳米羟基磷灰石悬浮液,进行后续实验。
3.MTT法检测不同浓度的分组以及多重对照组的细胞增殖情况,发现0.5%浓度的纳米羟基磷灰石悬浮液组对细胞增殖情况较其他组效果明显。在相同浓度(0.1%)下,纳米羟基磷灰石悬浮液组对细胞的增殖作用优于单独纳米羟基磷灰石组。
4.采用流式细胞术检测结果显示纳米羟基磷灰石悬浮液组可以减少处于G0/G1期的细胞数量,增加处于S期的细胞数量,使人牙周膜成纤维细胞在细胞增殖周期中很快完成G0/G1,期向s的过渡。
结论
1.纳米羟基磷灰石悬浮液对人牙周膜成纤维细胞的增殖有明显的促进作用。在细胞培养允许的渗透浓度内,浓度越高对细胞增殖的促进作用越明显,而且纳米羟基磷灰石对人牙周膜成纤维细胞增殖的促进作用在前24h最为明显。
2.纳米羟基磷灰石悬浮液作用于人牙周膜成纤维细胞,可以缩短细胞处于Go/G1,的时间,使较大量的细胞进入S期,进行有丝分裂,加速了HPDLCs的增殖速度。
3.通过本研究,进一步肯定了nHA运用于牙周软组织重建的可能,为nHA运用于牙周病的治疗提供了参考依据。
Objectives
1、Discussed the effect on periodontal and cell cycle of human ligament cells by hydroxyapatite suspension in the initial,so as to probe the application of nano-hydroxyapatite used in the treatment of disease.
2、In preparation for the future research on apoptosis of associated fibroblasts by the drug which was loaded with nano and mesoporous materials.
Method
1、Using tissue culture method to culture, identificate and passage human periodontal ligament fibroblasts, the 4th-6th fibroblasts were used for the experiment.
2、Prepared hydroxyapatite in the laboratory,used sodium carboxymethyl cellulose, sodium hexametaphosphate as dispersant. Compared the dispersion between the two dispersants,and compared the cell growth by cell culturing, choosed a suitable dispersant for the next researth.
3、Used the better dispersant which was choosed from the two above to disperse nano-hydroxyapatite.Prepared nano-hydroxyapatite suspension in 1%concentration.then diluted the concentration to 0.5%,0.25%,0.1%, 0.05% and 0.025%. Set some controls just as the blank, sigle hydroxyap-atite group in 0.1% concentration,sodium carboxymethyl cellulose in 0.1% concentration, mesoporous materials in 0.005% concentration and calcium hydroxide in 0.01% concentration.Then cultured the human periodontal ligament fibroblasts for 24 hours,48 huors and 72 huors in mdium with the groups above.And measured the proliferation of cells in each group with MTT to evaluate the proliferation effect of human periodontal ligament fibroblasts.
4^ Selected the suspension in 0.5% concentration as a new group,with the control groups together,human periodontal ligament fibroblasts were went on to culture for 48 huors.Then detected cell cycle distribution by flow cytometry to analysis the impacts of each group on culturing.
Result
1、Successfully cultured human periodontal ligament fibroblasts which identified by anti-vimentin and anti-keratin staining:anti-vimentin-positi-ve,and anti-keratin-negative.
2、Prepared nano-hydroxyapatite by chemical precipitation in laboratory, by testing, the diameter of nano-hydroxyapatite was from 40-60nm,which was required to this experiment. By comparing the sodium hexameta-phosphate and sodium carboxymethyl cellulose on the dispersion of nano-hydroxyapatite, sodium hexametaphosphate could be drawn to better in dispersing hydroxyapatite.However,it led to a large number of dead cells when nano-hydroxyapatite suspension which dispersed by sodium hexametaphosphate that was added in medium to culture human periodontal ligament fibroblasts.But the cells grow well in another group in which nano-hydroxyapatite was dispersed by sodium carboxymethyl cellulose. So sodium carboxymethyl cellulose was choosen to went on reseach as the dispersant we wanted to disperse the nano-hydroxyapatite suspension.
3、Cultured cells in different concentrations and multiple group which set for the control,then used MTT to detect proliferation of cells.The effect of proliferation was found significantly higher than other groups, when nano-hydroxyapatite was dispersed with sodium carboxymethyl cellulose in 0.5% concentration. At the same concentration(0.1%), the effect of cell proliferation was better in nano-hydroxyapatite suspension group than single nano-hydroxyapatite group.
4、The result by flow cytometry showed that it would reduce the number of cells in G0/G1 phase,when cell cultured in the group which nano-hydr-oxyapatite was dispersed in sodium carboxymethyl cellulose.At the same time, the number of cells in S phase would increase. It could be infered that human periodontal ligament fibroblasts would transite G0/G1 to S phase with the effect of nano-hydroxyapatite.
Conclusion
1、Dispersed in sodium carboxymethyl cellulose, nano-hydroxyapatite suspension had a good nano activity in its suspension,and it significantly promoted the proliferation of human periodontal ligament fibroblasts. Within the isotonic concentration, the higher the concentration was, the deeper the effection would be on the cell proliferation,and it led an obvious effect at the first 24 hours.
2、Dispersed in sodium carboxymethyl cellulose, nano-hydroxyapatite suspension acting on human periodontal ligament fibroblasts can shorten the G0/G1 phase time in human periodontal ligament fibroblasts' mitosis cycle, and made relatively large number of cells transite to S phase,for this it accelerated proliferation rate of human periodontal ligament fibroblasts
3、Through this study,It further affirmed that nano-hydroxyapatite can be used for reconstruction of periodontal soft tissue,and it provide a reference for nano-hydroxyapatite using in the treatment of periodontal disease.
引文
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[1]Edward L. Wolf. Nanophysics and Nanotechnology:An Introduction to Modern Concepts in Nanoscience. Weinheim:Wiley-VCH,2006:343.
[2]黄文,刘泉.颌骨囊肿摘除后即刻植入纳米羟基磷灰石的临床观察[J].华夏医学,2009,1(22)：56-57.
[3]Wei K, Li Y, Kim KO,et al.Fabrication of nano-hydroxyapatite on electrospun silk fibroin nanofiber and their effects in osteoblastic behavior[J].J Biomed Mater Res A. 2011,97(3):272-352.
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[6]王稚英,李世德,马晓凛等.个性化钛模板与纳米羟基磷灰石及自体骨修复兔上颌骨缺损[J].中国组织工程研究与临床康复2010,14(16)：2851-2852.
[7]陈鹏,刘冰,毛天球.活性纳米羟基磷灰石复合胶原/聚乳酸材料应用于牙槽嵴扩增的研究[J].口腔医学研究.2008,124(3)：282-284.
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[9]刘泉等.拔牙后牙槽窝即刻植入纳米羟基磷灰石的临床观察[J].柳州医学,2009,22(1)：27-28.
[10]刘冰,陈鹏,臧晓霞等.活性纳米羟基磷灰石复合胶原/聚乳酸材料对拔牙创早期愈合及牙槽嵴吸收影响的实验研究[J].口腔医学,2010,30(1)：38-41.
[11]董波,刘陆滨,刘玉杰.纳米羟基磷灰石修复慢性根尖周炎骨缺损的研究[J].黑龙江医药科学.2006,29(4)：103.
[12]闫永发,王春兰,范月静.纳米羟基磷灰石复合胶原植入术治疗牙周病骨缺损的疗效观察[J].中华临床医师杂志.2011,5(1)：239-240.
[13]孙波,李月玲,杨德龙纳米羟基磷灰石胶原骨植入治疗根分叉病变的临床研究[J].口腔医学.2010,30(6)：358-366.
[14]石铁,王泓杰,高秀秋.纳米羟基磷灰石及其复合材料对牙周膜细胞的影响[J].中国组织工程研究与临床康复.2009,13(3)：554-556.
[15]孙卫斌,吴亚菲,丁一,等.纳米羟基磷灰石对人牙周膜细胞增殖活性的影响.东南大学学报(自然科学版).2004,34(6)：802.
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[1]Edward L. Wolf. Nanophysics and Nanotechnology:An Introduction to Modern Concepts in Nanoscience. Weinheim:Wiley-VCH,2006:343.
[2]黄文,刘泉.颌骨囊肿摘除后即刻植入纳米羟基磷灰石的临床观察[J].华夏医学,2009,1(22)：56-57.
[3]Wei K, Li Y, Kim KO,et al.Fabrication of nano-hydroxyapatite on electrospun silk fibroin nanofiber and their effects in osteoblastic behavior[J].J Biomed Mater Res A. 2011,97(3):272-352.
[4]Kweon H, Lee KG, Chae CH,et al.Development of Nano-Hydroxyapatite Graft With Silk Fibroin Scaffold as a New Bone Substitute[J].J Oral Maxillofac Surg. 2011. [Epub ahead of print]
[5]李祖兵,胡图强,赵吉宏.混旋聚乳酸/纳米羟基磷灰石板在颧上颌骨骨折内固定中的应用[J].口腔医学研究.2004,20(3),228-228.
[6]王稚英,李世德,马晓凛等.个性化钛模板与纳米羟基磷灰石及自体骨修复兔上颌骨缺损[J].中国组织工程研究与临床康复2010,14(16)：2851-2852.
[7]陈鹏,刘冰,毛天球.活性纳米羟基磷灰石复合胶原/聚乳酸材料应用于牙槽嵴扩增的研究[J].口腔医学研究.2008,124(3)：282-284.
[8]He H, Yu J, Cao J,et al.Biocompatibility and Osteogenic Capacity of Periodontal Ligament Stem Cells on nHAC/PLA and HA/TCP Scaffolds[J].J Biomater Sci Polym Ed.2010. [Epub ahead of print]
[9]刘泉等.拔牙后牙槽窝即刻植入纳米羟基磷灰石的临床观察[J].柳州医学,2009,22(1)：27-28.
[10]刘冰,陈鹏,臧晓霞等.活性纳米羟基磷灰石复合胶原/聚乳酸材料对拔牙创早期愈合及牙槽嵴吸收影响的实验研究[J].口腔医学,2010,30(1)：38-41.
[11]董波,刘陆滨,刘玉杰.纳米羟基磷灰石修复慢性根尖周炎骨缺损的研究[J].黑龙江医药科学.2006,29(4)：103.
[12]闫永发,王春兰,范月静.纳米羟基磷灰石复合胶原植入术治疗牙周病骨缺损的疗效观察[J].中华临床医师杂志.2011,5(1)：239-240.
[13]孙波,李月玲,杨德龙纳米羟基磷灰石胶原骨植入治疗根分叉病变的临床研究[J].口腔医学.2010,30(6)：358-366.
[14]石铁,王泓杰,高秀秋.纳米羟基磷灰石及其复合材料对牙周膜细胞的影响[J].中国组织工程研究与临床康复.2009,13(3)：554-556.
[15]孙卫斌,吴亚菲,丁一,等.纳米羟基磷灰石对人牙周膜细胞增殖活性的影响.东南大学学报(自然科学版).2004,34(6)：802.
[16]滕立群,吕奎龙,孟祥才等.纳米羟基磷灰石抑制变形链球菌粘附的体外研究[J].黑龙江医药科学.2006,29(3)：20-21.
[17]李雪,周智,陈惠珍等.纳米羟基磷灰石对牙釉质再矿化影响的实验研究[J].牙体牙髓牙周病学杂志.2006,16(2)：67-68.
[18]Huang SB, Gao SS, Yu HY.Effect of nano-hydroxyapatite concentration on remineralization of initial enamel lesion in vitro[J].Biomed Mater.2009,4(3):104.
[19]Tschoppe P, Zandim DL, Martus P,et al. Enamel and dentine remineralization by nano-hydroxyapatite toothpastes[J]. J Dent.2011. [Epub ahead of print]
[20]谢民,赵立新,高成山.纳米羟基磷灰石对软化牙本质再矿化效果实验研究[J].贵州医药2009,33(1)：19-20.
[21]邱伟,时咏梅,徐进云.纳米羟基磷灰石直接盖髓诱导牙本质桥早期形成的可行性[J].中国组织工程研究与临床康复.2010,14(21)：3869-3872.
[22]谢民,黄敏,高成山.纳米羟基磷灰石盖髓材料临床应用研究[J].贵州医药2009,33(6)：547-548.
[23]周柳艳.纳米羟基磷灰石修补髓室底穿孔的临床疗效观察[J].口腔医学研究.2007,23(1)：115.
[24]杨青岭,李文婷,王健平等.壳聚糖/纳米羟基磷灰石治疗髓室底穿的实验研究[J].黑龙江医药.2007,30(2)：37.
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