纳米级钛颗粒对牙周组织细胞的影响
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摘要
目的通过研究纳米级钛颗粒对牙周膜细胞和牙槽骨细胞增值、分化的影响,探讨种植体脱落纳米级钛颗粒在种植体周围炎的发生、发展过程中的作用与机理。方法利用透射电子显微镜观察实验所用纳米级钛颗粒的大小与表面形貌;分离培养12月龄SPF级大鼠下颌第一磨牙牙周膜细胞和牙槽骨细胞,加入5×10~6粒/ml纳米级钛颗粒处理细胞,取该培养液为条件培养基,未加入纳米级钛颗粒的细胞和培养液作为对照组。培养2、4、6、8天后,倒置显微镜下观察细胞形态并计数;利用real-time PCR检测细胞培养24h后TNF-α和IL-1基因表达情况;当培养细胞发生接触抑制时,更换培养液并进行von Kossa染色后,观测钙化基质的沉积;利用real-time PCR检测细胞成骨标志基因的表达。分离大鼠胫骨和腓骨骨髓单核细胞,阳性对照加入20ng/ml MCSF和50ng/ml RANKL处理,实验组加入纳米级钛颗粒处理的或未处理的条件培养基,培养1周后,进行TRAP染色并计破骨细胞数。结果透射电子显微镜下,纳米级钛颗粒大小均一,直径约50～100nm,表面光滑;纳米级钛颗粒对牙周膜细胞和牙槽骨细胞增值具有微弱抑制作用,但无统计学差异(P﹥0.05);Real-time PCR结果显示,细胞培养24h后,实验组牙周膜细胞和牙槽骨细胞TNF-α和IL-1基因表达明显增高;von Kossa染色可见,纳米级钛颗粒处理的实验组牙周膜细胞和牙槽骨细胞成骨分化能力低于对照组;纳米级钛颗粒促进牙周膜细胞和牙槽骨细胞分泌RANKL(P<0.05),其中牙周膜细胞分泌较为显著;用含纳米级钛颗粒的条件培养基来处理大鼠骨髓单核细胞时,破骨细胞生成增多。结论纳米级钛颗粒对体外培养牙周膜细胞和牙槽骨细胞增值无明显作用,但是对其成骨分化有明显的抑制作用;同时促进牙周膜细胞和牙周骨细胞TNF-α、IL-1及RANKL的分泌;并可促进单核细胞分化为破骨细胞。
Objective To investigate the effect of titanium particles on the proliferation and differentiation of periodontal ligament and alveolar bone cells. Methods The cells were isolated from first molar and alveolar bone from 12-month-old SPF rats. The cells were treated with 5×10~6/ml yitanium particles. The cells without titanium particles were used as control. The cells were counted at day 2, 4, 6 and 8. The expression of TNF-α and IL-1 were measured by real time PCR and ELISA. To study osteogenic differentiation, the confluent cells were cultured in chemically defined osteogenic medium for 2 weeks, and the mineral matrix deposition was illustrated by van Kossastaining. Monocytes were isolated from rat bone marrows and propagated. The cells were treated with the conditional media of the periodontal ligament cells and alveolar bone cells treated with or without titanium particles. After one week, the differentiated osteoclast was stained by TRAP staining. Results Under the transmission electron microscopy, the particles showed a uniformed structure and diameter(50-100 nm). The particles slightly inhibited cell proliferation, but without statistical significance(P>0.05). Real-time PCR showed that, after 24 hours treatment,titanium particle stimulated the expression of inflammatory factors, TNF-α and IL-1. Titanium particles showed robust inhibitory effects on osteogenic differentiation. Interestingly, Titanium particles induced RANKL expression in both cell types, with more significant induction in periodontal ligament cells than in alveolar bone cells. Consistently, the conditional media from titanium treated periodontal ligament and alveolar bone cells enhanced monocytes osteoclastogenesis. Conclusion Titanium particles did not exert significant impact on proliferation of periodontal ligament and alveolar bone cells, but inhibited differentiation of both cell types. Titanium particles stimulated the expression of inflammatory factors and induced osteoclastogenesis.
Objective To investigate the effect of titanium particles on the proliferation and differentiation of periodontal ligament and alveolar bone cells. Methods The cells were isolated from first molar and alveolar bone from 12-month-old SPF rats. The cells were treated with 5×10~6/ml yitanium particles. The cells without titanium particles were used as control. The cells were counted at day 2, 4, 6 and 8. The expression of TNF-α and IL-1 were measured by real time PCR and ELISA. To study osteogenic differentiation, the confluent cells were cultured in chemically defined osteogenic medium for 2 weeks, and the mineral matrix deposition was illustrated by van Kossastaining. Monocytes were isolated from rat bone marrows and propagated. The cells were treated with the conditional media of the periodontal ligament cells and alveolar bone cells treated with or without titanium particles. After one week, the differentiated osteoclast was stained by TRAP staining. Results Under the transmission electron microscopy, the particles showed a uniformed structure and diameter(50-100 nm). The particles slightly inhibited cell proliferation, but without statistical significance(P>0.05). Real-time PCR showed that, after 24 hours treatment,titanium particle stimulated the expression of inflammatory factors, TNF-α and IL-1. Titanium particles showed robust inhibitory effects on osteogenic differentiation. Interestingly, Titanium particles induced RANKL expression in both cell types, with more significant induction in periodontal ligament cells than in alveolar bone cells. Consistently, the conditional media from titanium treated periodontal ligament and alveolar bone cells enhanced monocytes osteoclastogenesis. Conclusion Titanium particles did not exert significant impact on proliferation of periodontal ligament and alveolar bone cells, but inhibited differentiation of both cell types. Titanium particles stimulated the expression of inflammatory factors and induced osteoclastogenesis.
引文
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2 Thomas P,Summer B,Krenn V,et al.Allergy diagnostics in suspectedmetal implant intolerance.Orthopade,2013,42(8):602-606.
3 Pesce P,Canullo L,Grusovin MG,et al.Systematic review of some prosthetic riskfactors for periimplantitis.J Prosthet Dent,2015,114(3)346-350.
4王林红,樊立洁,谷志远.钛离子诱导骨吸收的现象及机制.国际口腔医学杂志,2009,36(4):441-443,447.
5 Cadosch D,Chan E,Gautschi OP,et al.Titanium induced osteoclastrecruitment and activation resulting in enhanced bone resorption:a human in vitro study.J Am Coll Surg,2008,207(3):S51.
6 Okazaki Y,Gotoh E,Manabe T,et al.Comparison of metal concentrations in rat tibia tissues with various metallic implants.Biomaterials,2004,25(28):5913-5920.
7 Hallab NJ,Jacobs JJ.Orthopedic implant fretting corrosion.Corros Rev,2003,21(2-3):183-214.
8 Saverino L,Granehi D,CiapettiG,et a1.Ion release in patients with metal-on-metal hip bearings in total joint replacement:a comparison with metal-on-polyethylene bearings.J Biomed Mater Res,2002,63(5):467-474.
9袁平.纳米级钛颗粒对异位骨化局部干预的基础研究.中南大学,2010.
10 Zhang Y,Yu W,Jiang X,et al.Analysis of the cytotoxicity of differentially sized titanium dioxide nanoparticles in murine MC3T3-E1 preosteoblasts.J Mater Sci Mater Med,2011,22(8):1933-1945.
11 Wei XC,Zhang XP,Lisa M,et al.Titanium particles stimulate COX-2 expression in synovial fibroblasts through an oxidative stressinduced,calpain-dependent,NF-κB pathway.Am J Physiol Cell Physiol,2009,297(2):C310-C320.
12李茂强,朱振安,刘凤翔,等.纳米级钛颗粒对大鼠成骨细胞增殖、分化及矿化功能的影响.中国组织工程研究,2009,13(26):5047-5051.
13 Peters K,Unger RE,Kirkpatrick CJ,et al.Effects of nano-scaled particles on endothelial cell function in vitro:studies on viability,proliferation and inflammation.J Mater Sci Mater Med.2004,15(4):321-325.
14杨书丰,赵建宁,薛孝威,等.钛合金微粒负荷成骨细胞对肿瘤坏死因子-α、白介素-1β和白介素-6的影响.中华骨质疏松和骨矿盐疾病杂志,2013,6(3):251-254.
15孟波,赖春花,吴王喜,等.钛离子对破骨细胞形成及功能的影响.广东医学,2012,3(11):1537-1538.
16 Badillo-Perona V,Cano-Sánchez J,Campo-Trapero J.Peri-implant bone mechanobiology.Review of the literature.Med Oral Patol Oral Cir Bucal,2011,16(5):e677-e681.
17 Vermes C,Roebuck KA,Chandrasekaran R,et al.Particulatewear debris activates protein tyrosine kinases and nuclear factor kappaB,which down-regulates type I collagen synthesis in human osteoblasts.J Bone Miner Res,2000,15(9):1756-1765.
18 Baeuerle PA.Pro-inflammatory signaling:last pieces in the NF-kappaB puzzle?Curr Biol,1998,8(1):R19-R22.
19陈建明,兰泽栋.骨形成蛋白对破骨细胞的调节作用.广东牙病防治,2008,16(4):189-190.
20郝奕霖,房付春,吴补领.微小RNA在人牙周膜来源细胞成骨分化中的作用.国际口腔医学杂志,2018,45(1):46-49.
21袁萍,李淑慧,赵璐,等.炎症微环境下人牙周膜干细胞的生物学特性.中国组织工程研究,2016,20(6):898-905.
2 Thomas P,Summer B,Krenn V,et al.Allergy diagnostics in suspectedmetal implant intolerance.Orthopade,2013,42(8):602-606.
3 Pesce P,Canullo L,Grusovin MG,et al.Systematic review of some prosthetic riskfactors for periimplantitis.J Prosthet Dent,2015,114(3)346-350.
4王林红,樊立洁,谷志远.钛离子诱导骨吸收的现象及机制.国际口腔医学杂志,2009,36(4):441-443,447.
5 Cadosch D,Chan E,Gautschi OP,et al.Titanium induced osteoclastrecruitment and activation resulting in enhanced bone resorption:a human in vitro study.J Am Coll Surg,2008,207(3):S51.
6 Okazaki Y,Gotoh E,Manabe T,et al.Comparison of metal concentrations in rat tibia tissues with various metallic implants.Biomaterials,2004,25(28):5913-5920.
7 Hallab NJ,Jacobs JJ.Orthopedic implant fretting corrosion.Corros Rev,2003,21(2-3):183-214.
8 Saverino L,Granehi D,CiapettiG,et a1.Ion release in patients with metal-on-metal hip bearings in total joint replacement:a comparison with metal-on-polyethylene bearings.J Biomed Mater Res,2002,63(5):467-474.
9袁平.纳米级钛颗粒对异位骨化局部干预的基础研究.中南大学,2010.
10 Zhang Y,Yu W,Jiang X,et al.Analysis of the cytotoxicity of differentially sized titanium dioxide nanoparticles in murine MC3T3-E1 preosteoblasts.J Mater Sci Mater Med,2011,22(8):1933-1945.
11 Wei XC,Zhang XP,Lisa M,et al.Titanium particles stimulate COX-2 expression in synovial fibroblasts through an oxidative stressinduced,calpain-dependent,NF-κB pathway.Am J Physiol Cell Physiol,2009,297(2):C310-C320.
12李茂强,朱振安,刘凤翔,等.纳米级钛颗粒对大鼠成骨细胞增殖、分化及矿化功能的影响.中国组织工程研究,2009,13(26):5047-5051.
13 Peters K,Unger RE,Kirkpatrick CJ,et al.Effects of nano-scaled particles on endothelial cell function in vitro:studies on viability,proliferation and inflammation.J Mater Sci Mater Med.2004,15(4):321-325.
14杨书丰,赵建宁,薛孝威,等.钛合金微粒负荷成骨细胞对肿瘤坏死因子-α、白介素-1β和白介素-6的影响.中华骨质疏松和骨矿盐疾病杂志,2013,6(3):251-254.
15孟波,赖春花,吴王喜,等.钛离子对破骨细胞形成及功能的影响.广东医学,2012,3(11):1537-1538.
16 Badillo-Perona V,Cano-Sánchez J,Campo-Trapero J.Peri-implant bone mechanobiology.Review of the literature.Med Oral Patol Oral Cir Bucal,2011,16(5):e677-e681.
17 Vermes C,Roebuck KA,Chandrasekaran R,et al.Particulatewear debris activates protein tyrosine kinases and nuclear factor kappaB,which down-regulates type I collagen synthesis in human osteoblasts.J Bone Miner Res,2000,15(9):1756-1765.
18 Baeuerle PA.Pro-inflammatory signaling:last pieces in the NF-kappaB puzzle?Curr Biol,1998,8(1):R19-R22.
19陈建明,兰泽栋.骨形成蛋白对破骨细胞的调节作用.广东牙病防治,2008,16(4):189-190.
20郝奕霖,房付春,吴补领.微小RNA在人牙周膜来源细胞成骨分化中的作用.国际口腔医学杂志,2018,45(1):46-49.
21袁萍,李淑慧,赵璐,等.炎症微环境下人牙周膜干细胞的生物学特性.中国组织工程研究,2016,20(6):898-905.