切换至 "中华医学电子期刊资源库"

中华口腔医学研究杂志(电子版) ›› 2016, Vol. 10 ›› Issue (01) : 30 -37. doi: 10.3877/cma.j.issn.1674-1366.2016.01.006

所属专题: 文献

基础研究

酸碱处理对多孔钛成骨细胞增殖和分化的影响
李雪莲1, 刘帅2, 简裕涛3, 孟跃中4, 赵克1,()   
  1. 1. 510055 广州,中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室;510080 广州,广东省牙颌系统修复重建技术与材料工程技术研究中心
    2. 510055 广州市越秀区口腔医院
    3. 510055 广州,中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室
    4. 510275 广州,中山大学材料科学与工程技术学院,广东省低碳化学与过程节能重点实验室
  • 收稿日期:2015-09-30 出版日期:2016-02-01
  • 通信作者: 赵克

Proliferation and differentiation of MC3T3-E1 cells on acid-alkali treated porous titanium

Xuelian Li1, Shuai Liu2, Yutao Jian3, Yuezhong Meng4, Ke Zhao1,()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China; Guangdong Engineering Research Center of Dental Technology and Materials for Restoration and Reconstruction, Guangzhou 510080, China
    2. Yuexiu Oral Hospital, Guangzhou 510055, China
    3. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
    4. School of Materials Science and Engineering, Sun Yat-sen University, The Key Laboratory of Low Carbon Chemistry & Energy Conservation of Guangdong Province, Guangzhou 510275, China
  • Received:2015-09-30 Published:2016-02-01
  • Corresponding author: Ke Zhao
  • About author:
    Corresponding author: Zhao Ke, Email:
引用本文:

李雪莲, 刘帅, 简裕涛, 孟跃中, 赵克. 酸碱处理对多孔钛成骨细胞增殖和分化的影响[J]. 中华口腔医学研究杂志(电子版), 2016, 10(01): 30-37.

Xuelian Li, Shuai Liu, Yutao Jian, Yuezhong Meng, Ke Zhao. Proliferation and differentiation of MC3T3-E1 cells on acid-alkali treated porous titanium[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2016, 10(01): 30-37.

目的

研究酸碱处理对多孔钛表面小鼠前成骨细胞MC3T3-E1增殖及分化的影响。

方法

粉末冶金法制备多孔钛,酸蚀处理后,NaOH浸泡不同时间,得到酸碱处理多孔钛(AAPT)。制备AAPT试件73个,未处理多孔钛(NTPT)试件41个和未处理致密钛(NTDT)试件30个。采用扫面电镜观察表面形貌,BCA法检测蛋白吸附能力,筛选适宜的碱处理时间。对于NTPT及AAPT组试件,采用表面接触角分析仪与拉曼光谱仪分析表面水接触角和化学组成。对于NTDT、NTPT及AAPT组试件,CCK-8法检测细胞增殖活性,实时荧光定量聚合酶链反应检测碱性磷酸酶(ALP)及骨钙素(OCN)基因相对表达量。采用独立样本t检验和单因素方差分析进行统计分析,LSD-t检验进行两两比较。

结果

12 h NaOH处理组(AA12PT)纳米结构最为清晰,且蛋白吸附量(0.367 mg/ml)最高(F= 400.835,P<0.05),因此采用该组进行后续实验。AA12PT组试件表面接触角(22.08°)显著小于NTPT组(93.7°),差异有统计学意义(F= 1.194,P<0.001)。AA12PT组试件可见钛酸氢钠特征散射峰。接种3、5、7 d后,NTDT组的细胞增殖活性均高于AA12PT组(F3 d= 245.517、F5 d= 102.442、F7 d= 119.047,P<0.001),AA12PT组均高于NTPT组(P3 d= 0.005、P5 d= 0.038、P7 d= 0.010)。接种7 d后,AA12PT与NTPT组的ALP基因和OCN基因相对表达量均高于NTDT组(FALP= 13.698,PALP-AA1 2PT< 0.001、PALP-NTPT= 0.002;FOCN= 175.859,POCN<0.001)。接种14 d后,NTDT组的ALP基因及OCN基因相对表达量均高于NTPT组(FALP= 33.691,PALP= 0.045;FOCN= 42.789,POCN= 0.044)。

结论

酸碱处理可显著提高多孔钛材料的蛋白吸附量,促进成骨细胞的增殖、分化。

Objective

To evaluate the proliferation and differentiation of MC3T3-E1 cells on acid-alkali treated porous titanium.

Methods

Porous titanium samples were fabricated by powder metallurgy process, then immersed in a mixed acid and NaOH solution in different periods to get acid-alkali-treated porous Ti (AAPT) . 73 AAPT samples, 41 non-treated porous Ti (NTPT) samples and 30 non-treated commercial dense titanium (NTDT) samples were made. The surface morphology was observed under a scanning electron microscopy. Protein adhesion was quantitatively analyzed by BCA method. The optimum treating time of alkali-treated in acid-alkali treatment was determined. For NTPT and AAPT, the contact angle and surface chemical structure were examined by an optical contact angle measuring device and a Raman spectrometer. CCK-8 and real-time fluorescence quantitative polymerase chain reaction were carried out to measure the proliferative activity and the gene expression of alkaline phosphatase (ALP) and osteocalcin (OCN) , respectively. The level of significance was determined by independent sample t test and an One-Way ANOVA followed by LSD-t test for a multiple comparison procedure.

Results

The scanning electron micrographs showed the nano structure of 12 h alkali treatment (AA12PT) was most distinct and obvious. And it had the highest protein adsorption (0.367 mg/ml) (F= 400.835, P<0.05) . So AA12PT was chosen in the subsequent experiments. The contact angle on the surfaces of NTPT (93.7°) exhibited a statistically significantly higher in comparison to the contact angle of AA12PT (22.08°) (F= 1.194, P<0.001) . The Raman spectroscopy indicated the presence of sodium hydrogen titanate on the surface of AA12PT. MC3T3-E1 cells got better proliferation rate on NTDT than AA12PT (F3 d= 245.517, F5 d= 102.442, F7 d= 119.047, P<0.001) at day 3, 5 and 7. And AA12PT enhanced cell proliferation significantly than NTPT (P3 d= 0.005, P5 d= 0.038, P7 d= 0.010) . At day 7, the gene expression of ALP and OCN of AA12PT and NTPT was higher than that of NTDT (FALP= 13.698, PALP-AA12PT<0.001, PALP-NTPT= 0.002; FOCN= 175.859, POCN<0.001) . At day 14, the gene expression of ALP and OCN of NTDT was higher than that of NTPT (FALP= 33.691, PALP= 0.045; FOCN= 42.789, POCN= 0.044) .

Conclusion

Acid-alkali treated porous titanium can significantly improve the amount of protein adsorption and promote proliferation and differentiation of osteoblasts.

表1 基因的引物序列
图1 扫描电镜下NTPT和AAPT试件的表面形貌
图2 各组多孔钛试件的蛋白吸附量
图3 AA12PT及NTPT试件的表面水接触角
图4 AA12PT及NTPT试件的拉曼光谱图
表2 不同时间MC3T3-E1细胞接种于各试件的增殖活性(A450±s
图5 MC3T3-E1细胞接种于各试件不同时间的增殖活性
图6 MC3T3-E1细胞接种于各试件不同时间的ALP基因相对表达量
图7 MC3T3-E1细胞接种于各试件不同时间的OCN基因相对表达量
[1]
Geetha M, Singh AK, Asokamani R,et al. Ti based biomaterials,the ultimate choice for orthopaedic implants - A review[J]. Prog Mater Sci,2009,54(3):397-425.
[2]
Chen Y, Feng B, Zhu Y,et al. Preparation and characterization of a novel porous titanium scaffold with 3D hierarchical porous structures[J]. J Mater Sci Mater Med,2011,22(4):839-844.
[3]
Chen D, Bertollo N, Lau A,et al. Osseointegration of porous titanium implants with and without electrochemically deposited DCPD coating in an ovine model[J]. J Orthop Surg Res,2011(6):56.
[4]
Rosa AL, Crippa GE, de Oliveira PT,et al. Human alveolar bone cell proliferation,expression of osteoblastic phenotype,and matrix mineralization on porous titanium produced by powder metallurgy[J]. Clin Oral Implants Res,2009,20(5):472-481.
[5]
刘帅,李湘霞,杨越,等.多孔纯钛孔隙率及孔隙尺寸对蛋白吸附及成骨细胞分化的影响[J/CD].中华口腔医学研究杂志:电子版,2015,9(3):193-199.
[6]
Yan WQ, Nakamura T, Kobayashi M,et al. Bonding of chemically treated titanium implants to bone[J]. Biomed Mater Res,1997,37(2):267-275.
[7]
Oliveira DP, Palmieri A, Carinci F,et al. Osteoblasts behavior on chemically treated commercially pure titanium surfaces[J]. J Biomed Mater Res A,2014,102(6):1816-1822.
[8]
叶琦,石新莹,曹姗姗,等.多孔钛孔隙率和孔隙尺寸对其力学性能及细胞相容性的影响[J].口腔材料器械杂志,2013,22(1):7-12.
[9]
Stevens MM, George JH. Exploring and engineering the cell surface interface[J]. Science,2005,310(5751):1135-1138.
[10]
Goto T, Yoshinari M, Kobayashi S,et al. The initial attachment and subsequent behavior of osteoblastic cells and oral epithelial cells on titanium[J]. Biomed Mater Eng,2004,14(4):537-544.
[11]
Zareidoost A, Yousefpour M, Ghaseme B,et al. The relationship of surface roughness and cell response of chemical surface modification of titanium[J]. J Mater Sci Mater Med,2012,23(6):1479-1488.
[12]
Feng B, Weng J, Yang BC,et al. Characterization of surface oxide films on titanium and adhesion of osteoblast[J]. Biomaterials,2003,24(25):4663-4670.
[13]
Rupp F, Scheideler L, Rehbein D,et al. Roughness induced dynamic changes of wettability of acid etched titanium implant modifications[J]. Biomaterials,2004,25(7-8):1429-1438.
[14]
Juodzbalys G, Sapragoniene M, Wennerberg A,et al. Titanium dental implant surface micromorphology optimization[J]. J Oral Implantol,2007,33(4):177-185.
[15]
Cho SA, Park KT. The removal torque of titanium screw inserted in rabbit tibia treated by dual acid etching[J]. Biomaterials,2003,24(20):3611-3617.
[16]
Wójciak-Stothard B, Curtis A, Monaghan W,et al. Guidance and activation of murine macrophages by nanometric scale topography[J]. Exp Cell Res,1996,223(2):426-435.
[17]
Dalby MJ, Gadegaard N, Herzyk P,et al. Nanomechanotransduction and interphase nuclear organization influence on genomic control[J]. J Cell Biochem,2007,102(5):1234-1244.
[18]
Zinger O, Zhao G, Schwartz Z,et al. Differential regulation of osteoblasts by substrate microstructural features[J]. Biomaterials,2005,26(14):1837-1847.
[19]
Buser D, Broggini N, Wieland M,et al. Enhanced bone apposition to a chemically modified SLA titanium surface[J]. J Dent Res,2004,83(7):529-533.
[20]
Wazen RM, Lefebvre LP, Baril E,et al. Initial evaluation of bone ingrowth into a novel porous titanium coating[J]. J Biomed Mater Res,2010,94(1):64-71.
[21]
Zhao C, Zhu X, Liang K,et al. Osteoinduction of porous titanium:a comparative study between acid-alkali and chemical-thermal treatments[J]. J Biomed Mater Res B Appl Biomater,2010,95(2):387-396.
[1] 凡军, 曹丽萍. 异鼠李素激活p38信号促进鼠间充质干细胞成骨分化[J]. 中华关节外科杂志(电子版), 2021, 15(04): 432-437.
[2] 陈玉书, 谢慧峰, 张燕红, 白波, 陈艺, 张姝江. 甲状旁腺激素在骨软骨组织中的应用进展[J]. 中华关节外科杂志(电子版), 2021, 15(02): 214-218.
[3] 刘鹏, 邓亚鹏, 曹国定, 高余, 封国超, 刘军, 甄平. 人工关节置换术后假体无菌性松动的研究进展[J]. 中华关节外科杂志(电子版), 2020, 14(03): 346-351.
[4] 杨萍, 许世敏, 李亮亮, 尹向云, 锡洪敏, 马丽丽, 李向红. 早产儿支气管肺发育不良合并代谢性骨病的影响因素[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(02): 202-211.
[5] 杜牧, 陈晓波, 宋福英, 刘子勤, 钱坤. CYP27B1基因突变所致维生素D依赖性佝偻病ⅠA型患儿临床特征与基因分析[J]. 中华妇幼临床医学杂志(电子版), 2022, 18(02): 175-184.
[6] 林伟斌, 朱聪, 洪海森, 黄国锋, 高明明, 吴进, 沙漠, 林灿斌, 陈娜娜, 张晓旭, 丁真奇. 体外周期性压应力对兔胫骨骨折愈合过程成骨与破骨细胞增殖分化能力的影响[J]. 中华损伤与修复杂志(电子版), 2021, 16(04): 289-300.
[7] 陈伟洋, 田俊, 韦曦. 硅离子在骨组织修复再生领域的作用[J]. 中华口腔医学研究杂志(电子版), 2021, 15(06): 375-381.
[8] 郑慧敏, 夏贤友, 刘梦, 姚晓雨, 隋磊. 整联蛋白在骨重建中的作用[J]. 中华口腔医学研究杂志(电子版), 2021, 15(02): 124-128.
[9] 马铭秀, 徐锋, 谢铠岭, 郭亚明, 卢潼辉, 戴朝六. 术前碱性磷酸酶-前白蛋白比值对肝细胞癌切除术预后的评估价值[J]. 中华普通外科学文献(电子版), 2023, 17(02): 99-103.
[10] 李蒋鹏, 徐婷, 杨发才, 赵芷藜, 刘小鹏, 何毅, 李敬东. APAR对肝细胞癌根治性切除术后患者预后的预测价值[J]. 中华肝脏外科手术学电子杂志, 2021, 10(03): 279-284.
[11] 彭汉书, 刘彬, 陈令斌, 刘融. 血清BALP、NTX在糖尿病足骨髓炎患者中的表达及意义[J]. 中华老年骨科与康复电子杂志, 2021, 07(06): 359-363.
[12] 遆云帆, 鲁经纬, 于多, 许斌, 赵建宁, 孙国静. 老年肺癌骨转移患者临床病理特征及预后生存分析[J]. 中华老年骨科与康复电子杂志, 2019, 05(05): 279-283.
[13] 高潮清, 刘勇, 鹿冬梅, 胡俊, 李慧, 周加军. 帕立骨化醇联合西那卡塞对尿毒症继发性甲状旁腺功能亢进患者微炎症状态、碱性磷酸酶及甲状旁腺激素水平的影响[J]. 中华临床医师杂志(电子版), 2022, 16(11): 1115-1119.
[14] 张林, 翟沛. 双氯芬酸钠联合骨肽注射液对老年膝关节骨性关节炎的治疗效果及安全性评估[J]. 中华临床医师杂志(电子版), 2021, 15(05): 335-340.
[15] 向茜, 张弦, 杨晓瑞, 栾艳, 徐静, 阮小荟, 李万碧. 个旧新诊断112例Graves'病患者骨代谢指标分析[J]. 中华肥胖与代谢病电子杂志, 2019, 05(03): 142-147.
阅读次数
全文


摘要