聚(L-谷氨酸)水凝胶介导羟基磷灰石的生物矿化

doi:10.7503/cjcu20180718

摘要/Abstract

摘要：

以1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC·HCl)为羧基活化剂, 己二酸二酰肼(ADH)为交联剂, 制备了生物活性聚(L-谷氨酸)(PLGA)水凝胶. 通过X射线衍射和扫描电子显微镜等表征了在不同浓度模拟体液(SBF)中羟基磷灰石(HA)的形成和生长. PLGA水凝胶的表面和内部均可观察到HA的形成和生长. 同时探讨了PLGA水凝胶矿化前后的力学性能. 将矿化前后PLGA水凝胶用于脂肪干细胞(ASCs)的培养, 研究其细胞相容性.

关键词: 聚(L-谷氨酸)水凝胶, 生物矿化, 羟基磷灰石, 骨再生

Abstract:

Bioactive poly(L-glutamic acid)(PLGA) hydrogels were prepared by using adipic acid dihydrazide(ADH) as a crosslinking agent. The formation and growth of hydroxyapatite(HA) in the simulated body fluid(SBF) of different concentrations were characterized by various techniques. PLGA is a kind of anionic poly(amino acid) with abundant carboxylic groups, which favors the enrichment of calcium ions and formation of HA. The growth of HA with soaking time was detected at the surface and interior of the PLGA hydrogels. The mechanical properties of the PLGA hydrogels before and after mineralization were studied in dynamic rheology experiments. Being cytocompatible, the mineralized PLGA hydrogels were successfully applied for cultivation of adipose-derived stem cells(ASCs). These results presented a method of preparing HA/PLGA composite hydrogels, which might be used as a promising biomaterial for bone tissue engineering.

Key words: Poly(L-glutamic acid) hydrogel, Biomimetic mineralization, Hydroxyapatite, Bone repair

中图分类号:

O636

TrendMD:

颜世峰, 王卫东, 任婕, 滕畅畅, 尹静波. 聚(L-谷氨酸)水凝胶介导羟基磷灰石的生物矿化. 高等学校化学学报, 2019, 40(4): 815.

YAN Shifeng,WANG Weidong,REN Jie,TENG Changchang,YIN Jingbo. Biomimetic Mineralization of Hydroxyapatite Mediated by Poly(L-glutamic acid) Hydrogels in Simulated Body Fluid^†. Chem. J. Chinese Universities, 2019, 40(4): 815.

图/表 13

Table 1 Comparation of the ion concentration(mmol/L) in human plasma, 1.0SBF and 1.5SBF

Sample	Ion concentration/(mmol·L^-1)
Sample	Na⁺	K⁺	Ca²⁺	Mg²⁺	HC $O 3 -$	Cl^-	HP $O 4 2 -$	S $O 4 2 -$
Plasma	142.0	5.0	2.5	1.5	27.0	103.0	1.0	0.5
1.0SBF	142.0	5.0	2.5	1.5	4.2	148.0	1.0	0.5
1.5SBF	213.1	7.5	3.8	2.3	6.3	221.9	1.5	0.8

Table 1 Comparation of the ion concentration(mmol/L) in human plasma, 1.0SBF and 1.5SBF

Sample	Ion concentration/(mmol·L^-1)
Sample	Na⁺	K⁺	Ca²⁺	Mg²⁺	HC $O 3 -$	Cl^-	HP $O 4 2 -$	S $O 4 2 -$
Plasma	142.0	5.0	2.5	1.5	27.0	103.0	1.0	0.5
1.0SBF	142.0	5.0	2.5	1.5	4.2	148.0	1.0	0.5
1.5SBF	213.1	7.5	3.8	2.3	6.3	221.9	1.5	0.8

Scheme 1 Schematic of PLGA crosslinking

Fig.1 Photographs of PLGA/ADH solution before(A) and after addition of EDC(B—D)(B) Investion; (C) lifting up; (D) picking up.

Fig.2 XRD patterns of PLGA hydrogels after soaking in 1.5SBF for different time(A) and comparison of XRD patterns of PLGA hydrogels after soaking in 1.0SBF and 1.5SBF(B)

Fig.3 FTIR spectra of PLGA(a), PLGA hydrogel before(b)and after mineralization(c)

Fig.4 Photographs of PLGA hydrogels before(A) and after soaking in 1.0SBF(B, B')/1.5SBF(C, C') for 14 d(B, C) and 28 d(B', C')

Fig.5 SEM images of the mineralized surface of PLGA hydrogels after soaking in 1.0SBF and 1.5SBF for different time (A—F) 2, 6, 10, 14, 21 and 28 d in 1.0SBF; (A'—F') 2, 6, 10, 14, 21 and 28 d in 1.5SBF; (G, H) different magnification in 1.5SBF for 14 d.

Fig.6 EDS spectrum of the cross-section of the hydrogel after soaking in 1.5SBF for 14 d

Fig.7 TGA curves of PLGA hydrogels after mineralization (A) PLGA hydrogels after soaking in 1.5SBF for different time; (B) comparison of TGA curves for PLGA hydrogels after soaking in 1.0SBF and 1.5SBF; (C) percentage of mass increase with immersion time for PLGA hydrogels after soaking in 1.0SBF and 1.5SBF.

Scheme 2 Schematic illustration of the HA mineralization induced by PLGA hydrogels Naka K(ed)

Fig.8 Rheological properties of PLGA hydrogels after mineralization(A) Storage modulus(G') or loss modulus(G″); (B) complex viscosity |η*| of PLGA hydrogels after soaking in 1.5SBF for different time.

Fig.9 SEM images of PLGA hydrogels before(A) and after soaking in 1.5SBF for 14 d(B) and the ASCs proliferated on PLGA hydrogel(C) and mineralized hydrogel for 3 d(D)

Fig.10 CLSM images of the ASCs-seeded PLGA hydrogels(A—C) and the ASCs-seeded mineralized hydrogel(D—F) at 1 d(A, D), 7 d(B, C) and 14 d(C, F) The living cells were stained with FDA(green) and the dead cell nuclei were stained with PI(red).

参考文献 52

[1]	Chirila T. V., Zainuddin., React. Funct.Polym.,2007, 67(3), 165—172
[2]	Song J., Saiz E., Bertozzi C. R.,J. Am. Chem.Soc.,2003, 125(5), 1236—1243
[3]	Webster T. J., Ergun C., Doremus R. H., Siegel R. W., Bizios R., Biomaterials,2000, 21(17), 1803—1810
[4]	Weiner S., Wagner H. D., Annu. Rev. Mater.Sci.,1998, 28, 271—298
[5]	Miyazaki T., Mukai J., Ishida E., Ohtsuki C., Biomed. Mater.Eng.,2013, 23(5), 339—347
[6]	Barrera D. A., Zylstra E., Lansbury P. T., Langer R., J. Am. Chem.Soc.,1993, 115(23), 11010—11011
[7]	Turco G., Marsich E., Bellomo F., Semeraro S., Donati I., Brun F., Grandoifo M., Accardo A., Paoletti S., Biomacromolecules,2009, 10(6), 1575—1583
[8]	Feng W., Feng S., Tang K., He X., Jing A., Liang G., J. Alloys Compd.,2017, 693, 482—489
[9]	Shu X., Shi Q., Feng J., Xie X., Chen Y., J. Mater.Sci.,2014, 49(22), 7742—7749
[10]	Luo Y., Lode A., Wu C., Chang J., Gelinsky M., ACS Appl. Mater.Interfaces,2015, 7(12), 6541—6549
[11]	Wang G., Zheng L., Zhao H., Miao J., Sun C., Liu H., Huang Z., Yu X., Wang J., Tao X., ACS Appl. Mater.Interfaces,2011, 3(5), 1692—1701
[12]	Gao X., Song J., Ji P., Zhang X., Li X., Xu X., Wang M., Zhang S., Deng Y., Deng F., Wei S., ACS Appl. Mater.Interfaces,2016, 8(5), 3499—3515
[13]	Yu S.H., Bio-inspired Crystal Growth by Synthetic Templates, In: Naka K. Ed., Biominer. Ⅱ, Springer, Berlin Heidelberg, 2006, 79—118
[14]	Hu C., Zhang L., Wei M., ACS Biomater. Sci.Eng.,2015, 1(8), 669—676
[15]	Zhu W., Lin J., Cai C., J. Mater.Chem.,2012, 22(9), 3939—3947
[16]	Turner J. S., Redpath G. T., Humphries J. E., Gonias S. L., Vandenberg S. R., Biochem.J.,1994, 297(1), 175—179
[17]	Ganss B., Kim R. H., Sodek J., Crit. Rev. Oral. Biol. Med.,1999, 10(1), 79—98
[18]	George A., Veis A., Chem.Rev.,2008, 108(11), 4670—4693
[19]	Coleman R. J., Jack K. S., Perrier S., Grøndahl L., Cryst. Growth Des.,2013, 13(10), 4252—4259
[20]	Liu Y., Li N., Qi Y., Dai L., Bryan T. E., Mao J., Pashley D. H., Tay F. R., Adv.Mater.,2011, 23(8), 975—980
[21]	Wang Y., Azaïs T., Robin M., Vallee A., Catania C., Legriel P., Pehau-Arnaudet G., Babonneau F., Giraud-Guille Nat. Mater.,2012, 11(8), 724—733
[22]	Li Y., Chen X., Fok A., Rodriguezcabello J. C., Aparicio C., ACS Appl. Mater.Interfaces,2015, 7(46), 25784—25792
[23]	Li C., Adv. Drug. Deliv.Rev., 2012, 54(5), 695—713
[24]	Song Z., Yin J., Luo K., Zheng Y., Yang Y., Li Q., Yan S., Chen X.,Macromol.Biosci.,2009, 9(3), 268—278
[25]	Qu X., Cui W., Yang F., Min C., Shen H., Bei J., Wang S., Biomaterials,2007, 28(1), 9—18
[26]	Yang Q., Peng J., Guo Q., Huang J., Zhang L., Yao J., Yang F., Wang S., Xu W., Wang A., Lu S., Biomaterials,2008, 29(15), 2378—2397
[27]	Cui L., Wu Y., Cen L., Zhou H., Yin S., Liu G., Liu W., Cao Y., Biomaterials,2009, 30(14), 2683—2693
[28]	Nakajima N., Ikada Y., Bioconjug.Chem.,1995, 6(1), 123—130
[29]	Tsai S., Hsieh C., Hsieh C., Wang D., Huang L., Lai J., Hsieh H., J. Appl. Polym.Sci.,2007, 105(4), 1774—1785
[30]	Nakajima N., Ikada Y., Bioconjug.Chem.,1995, 6(1), 123—130
[31]	Habibovic P., Barrère F., Blitterswijk C. A., Groot K. D., Layrolle P., J. Am. Ceram.Soc.,2004, 85(3), 517—522
[32]	Kokubo T., Hanakawa M., Kawashita M., Minoda M., Beppu T., Miyamoto T., Nakamura T., Biomaterials,2004, 25(18), 4485—4488
[33]	Ryu J., Ku S. H., Lee H., Park C., Adv. Funct.Mater.,2010, 20(13), 2132—2139
[34]	Dalby M. J., Gadegaard N., Tare R., Andar A., Riehle M. O., Herzyk P., Wilkinson C. D., Oreffo R. O.,Nat.Mater.,2007, 6(12), 997—1003
[35]	Webster T. J., Siegel R. W., Bizios R., Biomaterials,1999, 20(13), 1221—1227
[36]	Chou Y., Chiou W., Xu Y., Dunn J. C., Wu B. M., Biomaterials,2004, 25(22), 5323—5331
[37]	Yin J., Luo K., Chen X., Khutoryanskiv V. V., Carbohydr.Polym.,2006, 63(2), 238—244
[38]	Liu G., Zhao D., Tomsia A. P., Minor A. M., Song X., Saiz E., J. Am. Chem.Soc.,2009, 131(29), 9937—9939
[39]	Li P., Ohtsuki C., Kokubo T., Nakanishi K., Soga N., Nakamura T., Yamamuro T., J. Am. Ceram.Soc.,1992, 75(8), 2094—2097
[40]	Tanahashi M., Matsuda T.,J. Biomed. Mater.Res.,1997, 34(3), 305—315
[41]	Kawai T., Ohtsuki C., Kamitakahara M., Miyazaki T., Tanihara M., Sakaguchi Y., Konagaya S., Biomaterials,2004, 25(19), 4529—4534
[42]	Toworfe G. K., Composto R. J., Shapiro I. M., Ducheyne P., Biomaterials,2006, 27(4), 631—642
[43]	Kim C. W., Kim S. E., Kim Y. W., Lee H. J., Choi H. W., Chang J. H., Choi J., Kim K. J., Shim K. B., Jeong Y. K., Lee S. C., J. Mater.Res.,2009, 24(1), 50—57
[44]	Kawashita M., Nakao M., Minoda M., Kim H. M., Beppu T., Miyamoto K. T., Nakamura T., Biomaterials,2003, 24(14), 2477—2484
[45]	Jiao Y., Gyawali D., Stark J. M., Akcora P., Nair P., Tran R. T., Yang J., Soft Matter,2012, 8(5), 1499—1507
[46]	Tang Y., Du Y., Li Y., Wang X., Hu X., J. Biomed. Mater. Res. A,2009, 91A(4), 953—963
[47]	Heinemann S., Heinemann C., Jäger M., Neunzehn J., Wiesmann H. P., Hanke T., ACS Appl. Mater.Interfaces,2011, 3(11), 4323—4331
[48]	Fan H., Hu Y., Zhang C., Li X., Lv R., Qin L., Zhu R., Biomaterials,2006, 27(26), 4573—4580
[49]	Tan H., Ramirez C. M., Miljkovic N., Li H., Rubin J. P., Marra K. G., Biomaterials,2009, 30(36), 6844—6853
[50]	Yan S., Zhang K., Liu Z., Zhang X., Gan L., Cao B., Chen X., Cui L., Yin J., J. Mater. Chem.B,2013, 1(11), 1541—1553
[51]	Cao B., Yan S., Zhang K., Song Z., Cao T., Chen X., Cui L., Yin J., Macromol.Biosci.,2011, 11(7), 970—977
[52]	Chang C., Peng N., He M., Teramoto Y., Nishio Y., Zhang L., Carbohydr.Polym.,2013, 91(1), 7—13