原位沉淀法制备CS/nHA多孔复合支架及其性能

doi:10.7503/cjcu20180724

摘要/Abstract

摘要：

通过原位沉淀法和冷冻相分离技术得到含有钙磷前驱体(CaP)的初始多孔支架, 利用多孔支架表面原位生成的壳聚糖(CS)膜减缓NaOH溶液中OH^-离子的渗透速率, 以达到纳米羟基磷灰石(nHA)缓慢形成的目的, 从而制得nHA 分布均匀的CS/nHA多孔复合支架. 利用扫描电镜(SEM)和万能试验机研究复合支架的结构和性能, 发现nHA为针状结构, 长度为80200 nm, 宽度为2050 nm. 随着nHA含量的增加, 复合支架的孔隙率下降, 由(93.8±3.3)%降至(87.7±3.8)%, 压缩强度则逐渐提高, 由(0.5±0.09) MPa增加至(1.5±0.06) MPa. 当复合支架中nHA质量分数为25%时, 未发现nHA团聚现象, nHA均匀地分布于CS基体中. 通过红外光谱(FTIR)、 X射线衍射(XRD)及X射线光电子能谱(XPS)等分析推断, nHA与CS之间可能存在配位和氢键作用. 细胞实验结果表明, CS/nHA多孔复合支架具有良好的生物相容性, 细胞在支架内部贴壁黏附生长. CS/nHA多孔复合支架有望在骨组织工程领域具有良好的应用前景.

关键词: 原位沉淀法, 纳米羟基磷灰石, 壳聚糖, 骨组织工程, 多孔复合支架

Abstract:

The initial porous scaffold containing calcium phosphate precursors(CaP) was obtained by in situ precipitation and freezing phase separation techniques. The chitosan(CS) membranes generated in situ on the outer layer of porous scaffolds were used to slow down the penetration rate of OH^- ions in NaOH solution, so as to achieve the purpose of slow formation of nano-hydroxyapatite(nHA), and thus to prepare CS/nHA porous composite scaffolds with uniform distribution of nHA. The scanning electron microscopy(SEM) and universal testing machine were used to study the structure and properties of the composite scaffold. It was found that the nHA was acicular structure, with a length of 80―200 nm and a width of 20―50 nm. With the increase of nHA content, the porosity of the composite scaffold decreased from (93.8±3.3)% to (87.7±3.8)%, and the compressive strength gradually increased from (0.5±0.09) MPa to (1.5±0.06) MPa. No obvious agglomeration of nHA was found, and nHA was evenly distributed in the CS matrix when the content of nHA in the composite scaffold was 25%. It is concluded by infrared spectrometer(FTIR), X-ray diffractometer(XRD) and X-ray photoelectron spectrometer(XPS) analysis that there may be coordination and hydrogen bonding between nHA and CS. The results of cell experiments showed that the nHA/CS porous composite scaffold has good biocompatibility, and the cells adhered to the scaffold. It is expected that the CS/nHA porous composite scaffold has a good application prospect in the field of bone tissue engineering.

Key words: In situ precipitation, Nano-hydroxyapatite, Chitosan, Bone tissue engineering, Porous composite scaffold

中图分类号:

O636

TrendMD:

汪阮峰, 颜世峰, 胡圳, 尹静波. 原位沉淀法制备CS/nHA多孔复合支架及其性能. 高等学校化学学报, 2019, 40(5): 1080.

WANG Ruanfeng,YAN Shifeng,HU Zhen,YIN Jingbo. Preparation and Properties of CS/nHA Porous Composite Scaffold Based on In-situ Precipitation Method^†. Chem. J. Chinese Universities, 2019, 40(5): 1080.

图/表 13

Scheme 1 Preparation of CS/nHA composite scaffold

Fig.1 Pictures of scaffolds(A) Initial scaffold; (B) CS membrane-covered scaffold; (C) crosssection of CS membrane-covered scaffold;(D) CS/nHA composite scaffold.

Fig.2 FTIR spectra of composite scaffold

Fig.3 XRD patterns of composite scaffolds

Scheme 2 Schematic illustration of bonding between CS and nHA

Fig.4 XPS of CS scaffold(A, C) and CS/nHA25 scaffold(B, D) (A, B) N1s; (C, D) O1s.

Fig.5 TGA of composite scaffolds(A) and theoretical and measured values of nHA content in composite scaffolds(B)

Fig.6 SEM images of composite scaffolds(A) CS scaffold; (B) CS/nHA5 scaffold; (C) CS/nHA15 scaffold; (D) CS/nHA25 scaffold.

Fig.7 SEM image(A), EDS mappings(B, C) and high-magnification SEM image(D) and EDS analysis(inset) of CS/nHA25 scaffold

Fig.8 Porosity(A) and water uptake and retention levels(B) of composite scaffolds

Fig.9 Compression strength of composite scaffolds(A) and shape retention of composite scaffolds as a function of scaffold immersion time in SBF(B)

Fig.10 Live/dead staining images of rASCs cells cultured on CS/nHA25 scaffold for 1 d(A), 4 d(B), 7 d(C) and MTT assay for proliferation(D)

Fig.11 LSCM images of rASCs cells adhered to CS/nHA25 scaffold after 7 d of seeding Scaffold and cell nuclei stained blue(A) while the Actin filament stained red(B); (C) represented merged images of the two fluorochromes for the samples.

参考文献 51

[1]	Salgado A. J., Coutinho O. P., Reis R. L., Macromol. Biosci.,2004, 4(8), 743—765
[2]	Ou K. L., Hosseinkhani H., Int. J. Mol. Sci.,2014, 15(10), 17938—17962
[3]	Gao X., Song J., Ji P., ACS. Appl. Mater. Inter.,2016, 8(5), 3499—3515
[4]	Cong F., Zhang X., Savino K., Surf. Coat. Tech.,2016, 301, 13—19
[5]	Cucuruz A. T., Andronescu E., Ficai A., Int. J. Pharm.,2016, 510(2), 516—523
[6]	Dorozhkin S. V., Mater. Sci. Eng. C, 2015, 55, 272—326
[7]	Liu Y., Luo D., Wang T., Small,2016, 12(34), 4611—4632
[8]	Sun F., Zhou H., Lee J., Acta Biomater.,2011, 7(11), 3813—3828
[9]	Wang X., Bank R. A., Tekoppele J. M., J. Orthop. Res.,2001, 19(6), 1021—1026
[10]	Sionkowska A., Kaczmarek B., Lewandowska K., Int. J. Biol. Macromol.,2016, 89, 442—448
[11]	Chen L., Hu J., Ran J., Shen X., Int. J. Biol. Macromol.,2014, 65(5), 1—7
[12]	Li D., Sun H., Jiang L., ACS Appl. Mater. Interfaces,2014, 6(12), 9402—9410
[13]	Wang X., Li Y., Wei J., Biomaterials,2002, 23(24), 4787—4791
[14]	Borum L., Wilson O. C., Biomaterials,2003, 24(21), 3681—3688
[15]	Chen J., Nan K., Yin S., Colloid. Surface. B,2010, 81(2), 640—647
[16]	D'Elía N. L., Mathieu C., Hoemann C. D., Nanoscale,2015, 7(44), 18751—18762
[17]	Pu X. M., Yao Q. Q., Yang Y., Int. J. Biol. Macromol.,2012, 51(5), 868—873
[18]	Tavakol S., Nikpour M. R., Amani A., J. Nanopart. Res.,2013, 15(1), 1373
[19]	Maji S., Agarwal T., Das J., Carbohyd. Polym.,2018, 189, 115—125
[20]	Zhao F., Yin Y., Lu W. W., Biomaterials,2002, 23(15), 3227—3234
[21]	Koutsopoulos S., J. Biomed. Mater. Res. A, 2010, 62(4), 600—612
[22]	Venkatesan J., Kim S. K., J. Biomed. Nanotechnol.,2014, 10, 3124—3140
[23]	Yamaguchi I., Tokuchi K., Fukuzaki H., Koyama Y., Takakuda K., Monma H., Tanaka J., J Biomed. Mater. Res.,2015, 55(1), 20—27
[24]	Zhang Y., Venugopal J. R., El-Turki A., Ramakrishna S., Biomaterials,2008, 29, 4314—4322
[25]	Braem A., Chaudhari A., Cardoso M. V., Acta Biomater.,2014, 10(2), 986—995
[26]	Zhang J., Nie J., Zhang Q., J. Biomater. Sci. Polym. E,2014, 25(1),61—74
[27]	Theinhan W. W., Misra R. D., Acta Biomater.,2009, 5(4), 1182—1197
[28]	Depan D., Surya P. K. C. V., Acta Biomater.,2011, 7(5), 2163—2175
[29]	Moatary A., Teimouri A., Bagherzadeh M., Ceram. Int., 2017, 43(2), 1657—1688
[30]	Leong K. F., Cheah C. M., Chua C. K., Biomaterials,2003, 24(13), 2363—2378
[31]	Rusu V. M., Ng C. H., Wilke M., Adv. Sci. Technol.,2005, 53(26), 32—37
[32]	Shakir M., Zia I., Rehman A., Int. J. Biol. Macromol.,2018, 111, 903—916
[33]	Nikpour M. R., Rabiee S. M., Jahanshahi M., Composites Part B,2012, 43(4), 1881—1886
[34]	Sivakumar M., Manjubala I., Rao K. P., Carbohyd. Polym.,2002, 49(3), 281—288
[35]	Maji S., Agarwal T., Das J., Carbohyd. Polym.,2018, 189, 115—125
[36]	El-Sherbiny I. M., Yahia S., Messiery M. A., Int. J. Polym. Mater. Polym. Biomater.,2014, 63(4), 213—220
[37]	Li J., Dou Y., Yang J., Mater. Sci. Eng. C,2009, 29(4), 1207—1215
[38]	Hollister S. J., Nat. Mater., 2005, 4(7), 518—524
[39]	Vacanti J. P., Morse M. A., Mark S. W., Chinese J. Orthop. Traum.,2000, 23(1), 3—9
[40]	Yoshikawa T., Ohgushi H., Tamai S., J. Biomed. Mater. Res.,1996, 32(3), 481—492
[41]	Kong L., Yuan G., Cao W., J. Biomed. Mater. Res. P. A,2005, 75A(2), 275—282
[42]	Yan L. U., Zhu A., Wang W., Appl. Surf. Sci.,2010, 256(23), 7228—7233
[43]	Sun F., Zhou H., Lee J., Acta Biomater., 2011, 7, 3813—3828
[44]	Tavakol S., Nikpour M. R., Amani A., J. Nanop. Res.,2013, 15(1), 1373
[45]	Murugan R., Ramakrishna S., Biomaterials,2004, 25(17), 3829—3835
[46]	Hutmacher D. W., Biomaterials, 2000, 21(24), 2529—2543
[47]	Agrawal C. M., Ray R. B., J. Biomed. Mater. Res.,2001, 55(2), 141—150
[48]	Gunatillake P. A., Adhikari R., Eur. Cell Matter,2003, 5(5), 1—16
[49]	Kommareddy K. P., Lange C., Rumpler M., Biointerphases,2010, 5(2), 45—52
[50]	Lagonegro P., Trevisi G., Nasi L., Dent. Mater. J.,2018, 37(2), 278—285
[51]	Ma B., Zhang S., Liu F., ACS Appl. Mater. Int.,2017, 9(39), 33717—33727