人牙骨移植材料对小鼠单核巨噬细胞RAW264.7增殖分化的影响
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摘要
目的探讨人牙骨移植材料对巨噬细胞增殖分化以及形态的影响,了解人牙骨移植材料的生物相容性和细胞毒性。方法收集新鲜人离体牙,制备人牙骨移植材料,共聚焦显微镜下观察小鼠单核巨噬细胞RAW264.7与人牙骨移植材料共培养后对材料黏附情况。扫描电镜观察人牙骨移植材料、OSTEONⅡ人工骨植入物及未处理牙粉的形貌。配制不同成分浸提液,分4组:A组(含10%FBS的DMEM培养液)、B组(人牙骨移植材料)、C组(OSTEONⅡ人工骨植入物)、D组(未处理牙粉)。分别将4组浸提液与细胞共培养,倒置显微镜观察细胞形态变化定性判定细胞毒性,结合MTT法检测细胞增殖分化结果及细胞相对增殖率定量判定细胞毒性;通过锥虫蓝染色检测各组细胞活力;ELISA法检测炎性因子TNF-α和IL-6表达。结果扫描电镜观察示,人牙骨移植材料和OSTEONⅡ人工骨植入物的表面都具有均匀的孔状结构,而未经处理牙粉胶原纤维结构及脱矿牙本质层全层塌陷,无特定结构。共聚焦显微镜观察示,细胞在人牙骨移植材料上生长良好。细胞与浸提液共培养后,观察示A、B、C组细胞形态和数量正常,毒性反应分级均为0级,而D组均为3级。MTT检测示,B、C组各时间点细胞毒性均为0级或1级,提示材料合格;D组培养1 d时细胞毒性为2级,3、5、7 d均为4级,结合细胞形态分析,提示材料不合格。锥虫蓝染色示,各时间点A、B、C组细胞数量均显著高于D组,差异有统计学意义(P<0.05),A、B、C组间差异无统计学意义(P>0.05)。ELISA检测示,各时间点A、B、C组TNF-α和IL-6含量均显著低于D组,差异有统计学意义(P<0.05);A、B、C组间差异无统计学意义(P>0.05)。结论人牙骨移植材料与小鼠单核巨噬细胞共培养无细胞毒性,其浸提液对细胞增殖分化无明显影响,并不增加炎性因子表达,具有良好的生物相容性,有望用于临床骨缺损修复。
Objective To investigate the effect of human tooth bone graft materials on the proliferation,differentiation, and morphology of macrophages, and to understand the biocompatibility and cytotoxicity of human tooth bone graft materials. Methods Fresh human teeth were collected to prepare human tooth bone graft materials, the adhesion of mouse mononuclear macrophages RAW264.7 to human bone graft materials was observed under confocal microscopy. Scanning electron microscopy was used to observe the morphology of human tooth bone graft materials,OSTEONⅡ synthetic highly resorbable bone grafting materials, and untreated tooth powder(dental particles without preparation reagents). Different components of the extract were prepared in 4 groups: group A(DMEM medium containing 10% fetal bovine serum), group B(human tooth bone graft materials), group C(OSTEONⅡ synthetic highly resorbable bone grafting materials), group D(untreated tooth powder without preparation reagents). The 4 groups of extracts were co-cultured with the cells, and the cytotoxicity was qualitatively determined by observing the cell morphological changes by inverted microscope. The cell proliferation and differentiation results and cell relative proliferation rate were determined by MTT method to quantitatively determine cytotoxicity. The cell viability was detected by trypanosoma blue staining, and tumor necrosis factor α(TNF-α) and interleukin 6(IL-6) expressions were detected by ELISA. Results Scanning electron microscopy showed that the surface of the human tooth bone graft material and the OSTEONⅡ synthetic highly resorbable bone grafting materials had a uniform pore structure, while the untreated tooth particle collagen fiber structure and the demineralized dentin layer collapsed without specific structure.Confocal microscopy showed that the cells grew well on human tooth bone graft materials. After co-culture with the extract, the morphology and quantity of cells in groups A, B, and C were normal, and the toxic reaction grades were all grade 0, while group D was grade 3 reaction. MTT test showed that the cytotoxicity of groups B and C was grade 0 or 1 at each time point, indicating that the materials were qualified. The cytotoxicity was grade 2 in group D at 1 day after culture,and was grade 4 at 3, 5, and 7 days. Combined with cell morphology analysis, the materials were unqualified. The trypanosoma blue staining showed that the number of cells in groups A, B, and C was significantly higher than that in group D at each time point(P<0.05), but no significant difference was found among groups A, B, and C(P<0.05). ELISA test showed that the levels of TNF-α and IL-6 in groups A, B, and C were significantly lower than those in group D(P<0.05), but no significant difference was found among groups A, B, and C(P<0.05). Conclusion The human tooth bone graft materials is co-cultured with mice mononuclear macrophages without cytotoxicity. The extract has no significant effect on cell proliferation and differentiation, does not increase the expression of inflammatory factors, has good biocompatibility, and is expected to be used for clinical bone defect repair.
Objective To investigate the effect of human tooth bone graft materials on the proliferation,differentiation, and morphology of macrophages, and to understand the biocompatibility and cytotoxicity of human tooth bone graft materials. Methods Fresh human teeth were collected to prepare human tooth bone graft materials, the adhesion of mouse mononuclear macrophages RAW264.7 to human bone graft materials was observed under confocal microscopy. Scanning electron microscopy was used to observe the morphology of human tooth bone graft materials,OSTEONⅡ synthetic highly resorbable bone grafting materials, and untreated tooth powder(dental particles without preparation reagents). Different components of the extract were prepared in 4 groups: group A(DMEM medium containing 10% fetal bovine serum), group B(human tooth bone graft materials), group C(OSTEONⅡ synthetic highly resorbable bone grafting materials), group D(untreated tooth powder without preparation reagents). The 4 groups of extracts were co-cultured with the cells, and the cytotoxicity was qualitatively determined by observing the cell morphological changes by inverted microscope. The cell proliferation and differentiation results and cell relative proliferation rate were determined by MTT method to quantitatively determine cytotoxicity. The cell viability was detected by trypanosoma blue staining, and tumor necrosis factor α(TNF-α) and interleukin 6(IL-6) expressions were detected by ELISA. Results Scanning electron microscopy showed that the surface of the human tooth bone graft material and the OSTEONⅡ synthetic highly resorbable bone grafting materials had a uniform pore structure, while the untreated tooth particle collagen fiber structure and the demineralized dentin layer collapsed without specific structure.Confocal microscopy showed that the cells grew well on human tooth bone graft materials. After co-culture with the extract, the morphology and quantity of cells in groups A, B, and C were normal, and the toxic reaction grades were all grade 0, while group D was grade 3 reaction. MTT test showed that the cytotoxicity of groups B and C was grade 0 or 1 at each time point, indicating that the materials were qualified. The cytotoxicity was grade 2 in group D at 1 day after culture,and was grade 4 at 3, 5, and 7 days. Combined with cell morphology analysis, the materials were unqualified. The trypanosoma blue staining showed that the number of cells in groups A, B, and C was significantly higher than that in group D at each time point(P<0.05), but no significant difference was found among groups A, B, and C(P<0.05). ELISA test showed that the levels of TNF-α and IL-6 in groups A, B, and C were significantly lower than those in group D(P<0.05), but no significant difference was found among groups A, B, and C(P<0.05). Conclusion The human tooth bone graft materials is co-cultured with mice mononuclear macrophages without cytotoxicity. The extract has no significant effect on cell proliferation and differentiation, does not increase the expression of inflammatory factors, has good biocompatibility, and is expected to be used for clinical bone defect repair.
引文
1袁毅君,王廷璞,陈学梅,等.生物医用材料生物相容性评价研究进展.天水师范学院学报,2014,34(5):17-20.
2Kim YK,Yun PY,Um IW,et al.Alveolar ridge preservation of an extraction socket using autogenous tooth bone graft material for implant site development:prospective case series.J Adv Prosthodont,2014,6(6):521-527.
3Um IW,Kim YK,Mitsugi M.Demineralized dentin matrix scaffolds for alveolar bone engineering.J Indian Prosthodont Soc,2017,17(2):120-127.
4王媛,王稚英,王明.同种异体牙源性骨移植材料修复兔颅骨缺损的实验研究.中国医科大学学报,2016,45(12):1082-1085.
5于婷,曲守方,黄杰,等.医疗器械体外细胞毒性试验相关标准的比较及有关内容的商榷.国际检验医学杂志,2015,36(6):858-860.
6国丽荣.OSTEON修复犬下颌骨缺损的实验研究.沈阳:中国医科大学,2010.
7Su-Gwan K,Hak-Kyun K,Sung-Chul L.Combined implantation of particulate dentine,plaster of Paris,and a bone xenograft(OSTEONⅡ)for bone regeneration in rats.Journal of Cranio- Maxillofacial Surgery,2001,29(5):282-288.
8Jun SH,Ahn JS,Lee JI,et al.A prospective study on the effectiveness of newly developed autogenous tooth bone graft material for sinus bone graft procedure.J Adv Prosthodont,2014,6(6):528-538.
9Akazawa T,Murata M,Hino J,et al.Surface structure and biocompatibility of demineralized dentin matrix granules soaked in a simulated body fluid.Applied Surface Science,2012,262(12):51-55.
10熊航,谢志刚,鲍济波.脱矿牙本质基质的基本性能及制备.国际口腔医学杂志,2016,43(1):90-94.
11Murata M.Bone engineering using human demineralized dentin matrix and recombinant human BMP-2.J Hard Tissue Biology,2005,14(2):80-81.
12刘国林.脱矿牙本质基质诱导牙髓干细胞骨向分化研究.2014全国口腔生物医学学术年会暨“西湖国际”口腔医学高峰论坛论文集.杭州:中华口腔医学会口腔生物医学专业委员会,2014.
13卢贤欣,张东,谢迎,等.脱钙牙基质与成骨细胞的生物相容性.中国组织工程研究与临床康复,2009,13(47):9295-9298.
14Kim YK,Lee J,Um IW,et al.Tooth-derived bone graft material.J Korean Assoc Oral Maxillofac Surg,2013,39(3):103-111.
15Douard N,Leclerc L,Sarry G,et al.Impact of the chemical composition of poly-substituted hydroxyapatite particles on the in vitro pro-inflammatory response of macrophages.Biomed Microdevices,2016,18(2):27.
16Igeta K,Kuwamura Y,Horiuchi N,et al.Morphological and functional changes in RAW264 macrophage-like cells in response to a hydrated layer of carbonate-substituted hydroxyapatite.J Biomed Mater Res A,2017,105(4):1063-1070.
17Soltan M,Rohrer MD,Prasad HS.Monocytes:super cells for bone regeneration.Implant Dent,2012,21(1):13-20.
18Refai AK,Textor M,Brunette DM,et al.Effect of titanium surface topography on macrophage activation and secretion of proinflammatory cytokines and chemokines.J Biomed Mater Res A,2004,70(2):194-205.
19赖世翔,陈良娇,曹威,等.硅酸二钙对小鼠巨噬细胞系RAW264.7的促炎反应机制研究.口腔医学研究,2017,33(1):29-32.
20Morimoto S,Anada T,Honda Y,et al.Comparative study on in vitro biocompatibility of synthetic octacalcium phosphate and calcium phosphate ceramics used clinically.Biomed Mater,2012,7(4):045020.
21阮洪江,刘俊建,范存义,等.载银羟基磷灰石抗菌涂层体外抗菌性能及生物相容性研究.中国修复重建外科杂志,2009,23(2):226-230.
22乔玮,任晓琦,石浩,等.煅烧异种骨的生物相容性研究.中国修复重建外科杂志,2017,31(10):1250-1255.
23彭建强,易志新,武明鑫,等.氧化应激活化RAW264.7巨噬细胞对MC3T3-E1成骨细胞迁移、增殖及成骨基因表达影响的实验研究.中国修复重建外科杂志,2016,30(9):1146-1152.
24Bollati D,Morra M,Cassinelli C,et al.In vitro cytokine expression and in vivo healing and inflammatory response to a collagen-coated synthetic bone filler.Biomed Res Int,2016,2016:6427681.
25Douard N,Leclerc L,Sarry G,et al.Impact of the chemical composition of poly-substituted hydroxyapatite particles on the in vitro pro-inflammatory response of macrophages.BiomedMicrodevices,2016,18(2):27.
26Przekora A,Ginalska G.In vitro evaluation of the risk of inflammatory response after chitosan/HA and chitosan/β-1,3-glucan/HA bone scaffold implantation.Mater Sci Eng C Mater Biol Appl,2016,61:355-361.
27Gelse K,P?schl E,Aigner T.Collagens--structure,function,and biosynthesis.Adv Drug Deliv Rev,2003,55(12):1531-1546.
28Tang J,Saito T.Biocompatibility of novel typeⅠcollagen purified from tilapia fish scale:an in vitro comparative study.Biomed Res Int,2015,2015:139476.
29Nampo T,Watahiki J,Enomoto A,et al.A new method for alveolar bone repair using extracted teeth for the graft material.J Periodontol,2010,81(9):1264-1272.
30Kabir MA,Murata M,Akazawa T,et al.Evaluation of perforated demineralized dentin scaffold on bone regeneration in critical-size sheep iliac defects.Clin Oral Implants Res,2017,28(11):e227-e235.
31张建政,李亚非,骆洪涛,等.脱细胞保鲜异种骨移植免疫反应的实验研究.山西医科大学学报,2004,35(6):550-552.
32Chappard D,Fressonnet C,Genty C,et al.Fat in bone xenografts:importance of the purification procedures on cleanliness,wettability and biocompatibility.Biomaterials,1993,14(7):507-512.
33Moreau MF,Gallois Y,BasléMF,et al.Gamma irradiation of human bone allografts alters medullary lipids and releases toxic compounds for osteoblast-like cells.Biomaterials,2000,21(4):369-376.
2Kim YK,Yun PY,Um IW,et al.Alveolar ridge preservation of an extraction socket using autogenous tooth bone graft material for implant site development:prospective case series.J Adv Prosthodont,2014,6(6):521-527.
3Um IW,Kim YK,Mitsugi M.Demineralized dentin matrix scaffolds for alveolar bone engineering.J Indian Prosthodont Soc,2017,17(2):120-127.
4王媛,王稚英,王明.同种异体牙源性骨移植材料修复兔颅骨缺损的实验研究.中国医科大学学报,2016,45(12):1082-1085.
5于婷,曲守方,黄杰,等.医疗器械体外细胞毒性试验相关标准的比较及有关内容的商榷.国际检验医学杂志,2015,36(6):858-860.
6国丽荣.OSTEON修复犬下颌骨缺损的实验研究.沈阳:中国医科大学,2010.
7Su-Gwan K,Hak-Kyun K,Sung-Chul L.Combined implantation of particulate dentine,plaster of Paris,and a bone xenograft(OSTEONⅡ)for bone regeneration in rats.Journal of Cranio- Maxillofacial Surgery,2001,29(5):282-288.
8Jun SH,Ahn JS,Lee JI,et al.A prospective study on the effectiveness of newly developed autogenous tooth bone graft material for sinus bone graft procedure.J Adv Prosthodont,2014,6(6):528-538.
9Akazawa T,Murata M,Hino J,et al.Surface structure and biocompatibility of demineralized dentin matrix granules soaked in a simulated body fluid.Applied Surface Science,2012,262(12):51-55.
10熊航,谢志刚,鲍济波.脱矿牙本质基质的基本性能及制备.国际口腔医学杂志,2016,43(1):90-94.
11Murata M.Bone engineering using human demineralized dentin matrix and recombinant human BMP-2.J Hard Tissue Biology,2005,14(2):80-81.
12刘国林.脱矿牙本质基质诱导牙髓干细胞骨向分化研究.2014全国口腔生物医学学术年会暨“西湖国际”口腔医学高峰论坛论文集.杭州:中华口腔医学会口腔生物医学专业委员会,2014.
13卢贤欣,张东,谢迎,等.脱钙牙基质与成骨细胞的生物相容性.中国组织工程研究与临床康复,2009,13(47):9295-9298.
14Kim YK,Lee J,Um IW,et al.Tooth-derived bone graft material.J Korean Assoc Oral Maxillofac Surg,2013,39(3):103-111.
15Douard N,Leclerc L,Sarry G,et al.Impact of the chemical composition of poly-substituted hydroxyapatite particles on the in vitro pro-inflammatory response of macrophages.Biomed Microdevices,2016,18(2):27.
16Igeta K,Kuwamura Y,Horiuchi N,et al.Morphological and functional changes in RAW264 macrophage-like cells in response to a hydrated layer of carbonate-substituted hydroxyapatite.J Biomed Mater Res A,2017,105(4):1063-1070.
17Soltan M,Rohrer MD,Prasad HS.Monocytes:super cells for bone regeneration.Implant Dent,2012,21(1):13-20.
18Refai AK,Textor M,Brunette DM,et al.Effect of titanium surface topography on macrophage activation and secretion of proinflammatory cytokines and chemokines.J Biomed Mater Res A,2004,70(2):194-205.
19赖世翔,陈良娇,曹威,等.硅酸二钙对小鼠巨噬细胞系RAW264.7的促炎反应机制研究.口腔医学研究,2017,33(1):29-32.
20Morimoto S,Anada T,Honda Y,et al.Comparative study on in vitro biocompatibility of synthetic octacalcium phosphate and calcium phosphate ceramics used clinically.Biomed Mater,2012,7(4):045020.
21阮洪江,刘俊建,范存义,等.载银羟基磷灰石抗菌涂层体外抗菌性能及生物相容性研究.中国修复重建外科杂志,2009,23(2):226-230.
22乔玮,任晓琦,石浩,等.煅烧异种骨的生物相容性研究.中国修复重建外科杂志,2017,31(10):1250-1255.
23彭建强,易志新,武明鑫,等.氧化应激活化RAW264.7巨噬细胞对MC3T3-E1成骨细胞迁移、增殖及成骨基因表达影响的实验研究.中国修复重建外科杂志,2016,30(9):1146-1152.
24Bollati D,Morra M,Cassinelli C,et al.In vitro cytokine expression and in vivo healing and inflammatory response to a collagen-coated synthetic bone filler.Biomed Res Int,2016,2016:6427681.
25Douard N,Leclerc L,Sarry G,et al.Impact of the chemical composition of poly-substituted hydroxyapatite particles on the in vitro pro-inflammatory response of macrophages.BiomedMicrodevices,2016,18(2):27.
26Przekora A,Ginalska G.In vitro evaluation of the risk of inflammatory response after chitosan/HA and chitosan/β-1,3-glucan/HA bone scaffold implantation.Mater Sci Eng C Mater Biol Appl,2016,61:355-361.
27Gelse K,P?schl E,Aigner T.Collagens--structure,function,and biosynthesis.Adv Drug Deliv Rev,2003,55(12):1531-1546.
28Tang J,Saito T.Biocompatibility of novel typeⅠcollagen purified from tilapia fish scale:an in vitro comparative study.Biomed Res Int,2015,2015:139476.
29Nampo T,Watahiki J,Enomoto A,et al.A new method for alveolar bone repair using extracted teeth for the graft material.J Periodontol,2010,81(9):1264-1272.
30Kabir MA,Murata M,Akazawa T,et al.Evaluation of perforated demineralized dentin scaffold on bone regeneration in critical-size sheep iliac defects.Clin Oral Implants Res,2017,28(11):e227-e235.
31张建政,李亚非,骆洪涛,等.脱细胞保鲜异种骨移植免疫反应的实验研究.山西医科大学学报,2004,35(6):550-552.
32Chappard D,Fressonnet C,Genty C,et al.Fat in bone xenografts:importance of the purification procedures on cleanliness,wettability and biocompatibility.Biomaterials,1993,14(7):507-512.
33Moreau MF,Gallois Y,BasléMF,et al.Gamma irradiation of human bone allografts alters medullary lipids and releases toxic compounds for osteoblast-like cells.Biomaterials,2000,21(4):369-376.