两种富血小板纤维蛋白对人牙龈成纤维细胞增殖活性影响的比较
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摘要
背景:自体血液制成的富血小板纤维蛋白在促进软组织再生修复方面成为研究热点。目的:对比2种富血小板纤维蛋白对人牙龈成纤维细胞生长速度的影响,为临床中选择口腔软组织再生修复材料提供参考。方法:阻生牙拔除同期取患牙周围健康龈瓣(8例),组织块培养法获取人牙龈成纤维细胞并进行鉴定。抽取对应患者静脉血分别制成改良型富血小板纤维蛋白、富血小板纤维蛋白,8例样本均取第5代细胞分改良型富血小板纤维蛋白、富血小板纤维蛋白、空白组3组进行培养,于第1,3,5及7天,CCK-8法检测细胞增殖活性。结果与结论:(1)培养的人牙龈成纤维细胞Vimentin染色阳性,Cytokeratin染色阴性;(2)成功获得2种富血小板纤维蛋白凝胶;与富血小板纤维蛋白相比,改良型富血小板纤维蛋白颜色较深,膜更厚,韧性更好;(3)CCK-8结果显示,第1天,3组细胞增殖活性无显著差异(P> 0.05);第3天,改良型富血小板纤维蛋白和富血小板纤维蛋白组的细胞增殖活性显著高于空白组(P <0.05);第5和7天,细胞增殖活性表现为改良型富血小板纤维蛋白组>富血小板纤维蛋白组>空白组(P <0.01,P <0.05);(4)结果提示,改良型富血小板纤维蛋白、富血小板纤维蛋白均能提高成纤维细胞增殖活性,且改良型富血小板纤维蛋白促细胞增殖效果明显优于富血小板纤维蛋白。
BACKGROUND: Platelet-rich fibrin(PRF) prepared by autologous blood has become the hotspot in regeneration and repair of soft tissues. OBJECTIVE: To compare the effect of two kinds of PRFs on the proliferation activity of human gingival fibroblasts(HGFs), so as to provide reference for the selection of oral soft tissue regeneration and repair materials in the clinic. METHODS: Healthy gingival flaps from eight patients were collected when mandibular impacted teeth were removed. HGFs were obtained using tissue explant method, and then identified. The venous blood of the corresponding patients was drawn and made into advanced PRF and PRF, respectively, and passage 5 HGFs were divided into advanced PRF, PRF and blank groups for culture. The cell proliferation activity was detected by cell counting-kit 8 assay at 1, 3, 5, and 7 days. RESULTS AND CONCLUSION: The HGFs were positive for Vimentin staining and negative for Cytokeratin staining. Two kinds of PRFs were prepared successfully. The advanced PRF exhibited the deeper color, more thick membrane and better flexibility compared with the general PRF. Cell counting-kit 8 assay results revealed that at 1 day after culture, there was no significant difference in the proliferation activity among groups(P > 0.05). At 3 days, the cell proliferation activity in the advanced PRF and PRF groups was significantly higher than that in the blank group(P < 0.05). At 5 and 7 days, the order of cell proliferation activity was as follows: advanced PRF group > PRF group > blank group(P < 0.01, P < 0.05). To conclude, both advanced PRF and PRF can increase the proliferation activity of HGFs, especially the former.
BACKGROUND: Platelet-rich fibrin(PRF) prepared by autologous blood has become the hotspot in regeneration and repair of soft tissues. OBJECTIVE: To compare the effect of two kinds of PRFs on the proliferation activity of human gingival fibroblasts(HGFs), so as to provide reference for the selection of oral soft tissue regeneration and repair materials in the clinic. METHODS: Healthy gingival flaps from eight patients were collected when mandibular impacted teeth were removed. HGFs were obtained using tissue explant method, and then identified. The venous blood of the corresponding patients was drawn and made into advanced PRF and PRF, respectively, and passage 5 HGFs were divided into advanced PRF, PRF and blank groups for culture. The cell proliferation activity was detected by cell counting-kit 8 assay at 1, 3, 5, and 7 days. RESULTS AND CONCLUSION: The HGFs were positive for Vimentin staining and negative for Cytokeratin staining. Two kinds of PRFs were prepared successfully. The advanced PRF exhibited the deeper color, more thick membrane and better flexibility compared with the general PRF. Cell counting-kit 8 assay results revealed that at 1 day after culture, there was no significant difference in the proliferation activity among groups(P > 0.05). At 3 days, the cell proliferation activity in the advanced PRF and PRF groups was significantly higher than that in the blank group(P < 0.05). At 5 and 7 days, the order of cell proliferation activity was as follows: advanced PRF group > PRF group > blank group(P < 0.01, P < 0.05). To conclude, both advanced PRF and PRF can increase the proliferation activity of HGFs, especially the former.
引文
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[10]Bishara M,Kurtzman GM,Khan W,et al.Soft-tissue grafting techniques associated with immediate implant placement.Compend Contin Educ Dent.2018;39(2):e1-4.
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[14]Xu QC,Wang ZG,Ji QX,et al.Systemically transplanted human gingiva-derived mesenchymal stem cells contributing to bone tissue regeneration.Int J Clin Exp Pathol.2014;7(8):4922-4929.
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[16]杨琴秋.富血小板纤维蛋白诱导口腔软组织缺损修复再生的实验研究[D].成都:四川医科大学,2015.
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[18]Dohan Ehrenfest DM,Rasmusson L.Classification of platelet concentrates:from pure platelet-rich plasma(P-PRP)to leucocyte-and platelet-rich fibrin(L-PRF).Trends Biotechnol.2009;27(3):158-167.
[19]Dohan Ehrenfest D M.How to optimize the preparation of leukocyte-and platelet-rich fibrin(L-PRF,Choukroun's technique)clots and membranes:introducing the PRF Box.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2010;110(3):278-280.
[20]练轶,毛俊丽,孙嵩,等.改良型富血小板纤维蛋白与富血小板纤维蛋白的成分及超微结构观察[J].西南国防医药.2017;27(6):548-551.
[21]Dohan Ehrenfest DM,Diss A,Odin G,et al.In vitro effects of Choukroun's platelet-rich fibrin on human gingival fibroblasts,dermal prekeratinocytes,preadipocytes,and maxillofacial osteoblasts in primary cultures.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2009;108(3):341-352.
[22]Fujioka-Kobayashi M,Miron RJ,Hernandez M,et al.Optimized platelet rich fibrin with the low speed concept:growth factor release,biocompatibility and cellular response.J Periodontol.2016;88(1):112-121.
[23]Dohan Ehrenfest DM,Pinto NR,Pereda A,et al.The impact of the centrifuge characteristics and centrifugation protocols on the cells,growth factors,and fibrin architecture of a leukocyte-and platelet-rich fibrin(L-PRF)clot and membrane.Platelets.2018;29(2):171-184.
[24]Kobayashi E,Flückiger L,Fujioka-Kobayashi M,et al.Comparative release of growth factors from PRP,PRF,and advanced-PRF.Clin Oral Investig.2016;20(9):2353-2360.
[25]Liu Y,Kalén A,Risto O,et al.Fibroblast proliferation due to exposure to a platelet concentrate in vitro is pH dependent.Wound Repair Regen.2002;10(5):336-340.
[26]Graziani F,Ivanovski S,Cei S,et al.The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts.Clin Oral Implants Res.2006;17(2):212-219.
[27]Goto H,Matsuyama T,Miyamoto M,et al.Platelet-rich plasma/osteoblasts complex induces bone formation via osteoblastic differentiation following subcutaneous transplantation.J Periodontal Res.2006;41(5):455-62.
[28]Slapnicka J,Fassmann A,Strasak L,et al.Effects of activated and nonactivated platelet-rich plasma on proliferation of human osteoblasts in vitro.J Oral Maxillofac Surg.2008;66(2):297-301.
[29]何婷婷.两种富血小板纤维蛋白的降解特性研究[D].泸州:西南医科大学,2017.
[2]Miron RJ,Zucchelli G,Pikos MA,et al.Use of platelet-rich fibrin in regenerative dentistry:a systematic review.Clin Oral Investig.2017;21(6):1913-1927.
[3]Vahabi S,Vaziri S,Torshabi M.Effects of plasma rich in growth factors and platelet-rich fibrin on proliferation and viability of human gingival fibroblasts.J Dent(Tehran).2015;12(7):504-512.
[4]Polimeni G,Xiropaidis AV,Wikesj?UME.Biology and principles of periodontal wound healing/regeneration.Periodontology.2006;41(1):30.
[5]Garg AK.The use of platelet-rich plasma to enhance the success of bone grafts around dental implants.Dental Implantology Update.2000;11(3):17.
[6]Landesberg R,Moses M.Risks of using platelet rich plasma gel.J Oral Maxillofac Surg.1998;56(9):1116-1117.
[7]Choukroun J,Adda F,Schoefer C,et al.An opportunity in perio-implantology:The PRF.Implantodontie.2000;42:55-62.
[8]Rodella LF,Favero G,Boninsegna R,et al.Growth factors,CD34 positive cells,and fibrin network analysis in concentrated growth factors fraction.Microsc Res Tech.2011;74(8):772-777.
[9]Gupta S,Banthia R,Singh P,et al.Clinical evaluation and comparison of the efficacy of coronally advanced flap alone and in combination with platelet rich fibrin membrane in the treatment of Miller Class I and II gingival recessions.Contemp Clin Dent.2015;6(2):153-160.
[10]Bishara M,Kurtzman GM,Khan W,et al.Soft-tissue grafting techniques associated with immediate implant placement.Compend Contin Educ Dent.2018;39(2):e1-4.
[11]Ocak H,Kutuk N,Demetoglu U,et al.Comparison of bovine bone-autogenic bone mixture versus platelet-rich fibrin for maxillary sinus grafting:histologic and histomorphologic study.J Oral Implantol.2017;43(3):194-201.
[12]Ghanaati S,Booms P,Orlowska A,et al.Advanced platelet-rich fibrin:a new concept for cell-based tissue engineering by means of inflammatory cells.J Oral Implantol.2014;40(6):679-689.
[13]Taoufik K,Mavrogonatou E,Eliades T,et al.Effect of blue light on the proliferation of human gingival fibroblasts.Dental Materials.2008;24(7):895-900.
[14]Xu QC,Wang ZG,Ji QX,et al.Systemically transplanted human gingiva-derived mesenchymal stem cells contributing to bone tissue regeneration.Int J Clin Exp Pathol.2014;7(8):4922-4929.
[15]Soundara Rajan T,Scionti D,Diomede F,et al.Prolonged expansion induces spontaneous neural progenitor differentiation from human gingiva-derived mesenchymal stem cells.Cell Reprogram.2017;19(6):389-401.
[16]杨琴秋.富血小板纤维蛋白诱导口腔软组织缺损修复再生的实验研究[D].成都:四川医科大学,2015.
[17]Eren G.Platelet-rich fibrin in the treatment of localized gingival recessions:a split-mouth randomized clinical trial.Clin Oral Investig.2014;18(8):1941-1948.
[18]Dohan Ehrenfest DM,Rasmusson L.Classification of platelet concentrates:from pure platelet-rich plasma(P-PRP)to leucocyte-and platelet-rich fibrin(L-PRF).Trends Biotechnol.2009;27(3):158-167.
[19]Dohan Ehrenfest D M.How to optimize the preparation of leukocyte-and platelet-rich fibrin(L-PRF,Choukroun's technique)clots and membranes:introducing the PRF Box.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2010;110(3):278-280.
[20]练轶,毛俊丽,孙嵩,等.改良型富血小板纤维蛋白与富血小板纤维蛋白的成分及超微结构观察[J].西南国防医药.2017;27(6):548-551.
[21]Dohan Ehrenfest DM,Diss A,Odin G,et al.In vitro effects of Choukroun's platelet-rich fibrin on human gingival fibroblasts,dermal prekeratinocytes,preadipocytes,and maxillofacial osteoblasts in primary cultures.Oral Surg Oral Med Oral Pathol Oral Radiol Endod.2009;108(3):341-352.
[22]Fujioka-Kobayashi M,Miron RJ,Hernandez M,et al.Optimized platelet rich fibrin with the low speed concept:growth factor release,biocompatibility and cellular response.J Periodontol.2016;88(1):112-121.
[23]Dohan Ehrenfest DM,Pinto NR,Pereda A,et al.The impact of the centrifuge characteristics and centrifugation protocols on the cells,growth factors,and fibrin architecture of a leukocyte-and platelet-rich fibrin(L-PRF)clot and membrane.Platelets.2018;29(2):171-184.
[24]Kobayashi E,Flückiger L,Fujioka-Kobayashi M,et al.Comparative release of growth factors from PRP,PRF,and advanced-PRF.Clin Oral Investig.2016;20(9):2353-2360.
[25]Liu Y,Kalén A,Risto O,et al.Fibroblast proliferation due to exposure to a platelet concentrate in vitro is pH dependent.Wound Repair Regen.2002;10(5):336-340.
[26]Graziani F,Ivanovski S,Cei S,et al.The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts.Clin Oral Implants Res.2006;17(2):212-219.
[27]Goto H,Matsuyama T,Miyamoto M,et al.Platelet-rich plasma/osteoblasts complex induces bone formation via osteoblastic differentiation following subcutaneous transplantation.J Periodontal Res.2006;41(5):455-62.
[28]Slapnicka J,Fassmann A,Strasak L,et al.Effects of activated and nonactivated platelet-rich plasma on proliferation of human osteoblasts in vitro.J Oral Maxillofac Surg.2008;66(2):297-301.
[29]何婷婷.两种富血小板纤维蛋白的降解特性研究[D].泸州:西南医科大学,2017.