与胶原特异性结合的BMP2模拟肽/PLGA 3D打印复合支架的制备及成骨诱导活性

doi:10.7503/cjcu20190145

摘要/Abstract

摘要：

将胶原绑定结构域(CBD)多肽序列与骨形态发生蛋白2模拟肽(BMP2-MP)序列连接制备具有胶原绑定能力的CBD-BMP2-MP, 再将CBD-BMP2-MP与聚丙交酯-乙交酯/胶原(PLGA/COL)3D打印支架相结合, 以支架表面的胶原成分为媒介, 将CBD-BMP2-MP更有效地固定于骨修复材料上, 达到对其进行改性的目的. 利用扫描电子显微镜(SEM)、电子万能试验机和接触角测量仪对复合支架表面形貌、力学强度和亲水性等材料学性能进行评价. 用荧光成像法评测 CBD-BMP2-MP及BMP2-MP与支架材料的结合能力. 在各组支架材料表面接种MC3T3-E1细胞进行体外培养, 采用CCK-8、鬼笔环肽荧光染色、茜素红染色及qPCR综合评价细胞在材料表面的黏附、增殖和成骨分化等细胞行为, 研究CBD-BMP2-MP修饰的3D多孔PLGA/COL复合支架的生物学性能. 研究结果表明, 利用3D打印技术制备的多孔支架具有形貌可控的孔隙结构, 为细胞生长创造更有利的细胞微环境, 支架表面胶原成分的加入提高了支架材料的亲水性, 同时对支架材料本身的力学性能无任何影响, 提高了复合支架本身的生物相容性. 与普通BMP2-MP相比, CBD-BMP2-MP具有更好的胶原绑定能力, 与复合支架的结合更稳定, 提高了PLGA/COL复合支架对BMP2-MP的负载能力. 支架表面负载CBD-BMP2-MP后具有极强的促细胞成骨分化能力. MC3T3-E1细胞表现出更高的钙沉积能力, 并且成骨分化相关基因Runx2, ALP, COL-I及OPN等水平也有了明显提升. 表明CBD-BMP2-MP多孔复合支架具有良好的生物相容性和成骨诱导活性, 在骨组织修复领域具有良好的应用前景.

关键词: 胶原特异结合性, 骨形态发生蛋白2模拟肽, 聚丙交酯-乙交酯, 3D打印支架, 成骨诱导活性

Abstract:

Collagen binding domain(CBD) was conjugated to bone morphogenetic protein-2 mimetic peptide(BMP2-MP) to prepare collagen-binding BMP2-MP(CBD-BMP2-MP), then, CBD-BMP2-MP was combined with PLGA/collagen(PLGA/COL) composite scaffold for the surface modification of scaffold. SEM, electronic universal testing machine and contact angle measuring instrument were used to evaluate the surface morphology, mechanical strength, hydrophilicity and other material properties of PLGA/COL scaffolds. The binding abilities of CBD-BMP2-MP and BMP2-MP to scaffold materials were evaluated by immunofluorescence analysis. Subsequently, MC3T3-E1 cells were grown on different scaffolds, and cell proliferation, adhesion and osteogenic differentiation on different scaffold were evaluated by CCK-8, fluorescence staining, ARS and qPCR. The results show that the 3D porous scaffold has regular and controllable pore structure, which can create more favorable cell microenvironment for cell growth. The addition of collagen on the scaffold surface improved the hydrophilicity of material, and had no effect on the mechanical properties of material itself, which greatly improved the biocompatibility of the 3D scaffold. Compared with commercial BMP2-MP, CBD-BMP2-MP had better collagen binding ability, which improves the combining capacity of growth factor and PLGA/COL scaffold. The scaffold surface loaded with CBD-BMP2-MP has a strong ability to promote osteoblastic differentiation. MC3T3-E1 cells showed higher calcium deposition capacity, and the levels of osteogenic related gens such as COI-1, Runx2, ALP and OPN were also significantly increased. The results indicated that CBD-BMP2-MP modified 3D porous PLGA/COL scaffold has good biocompatibility and osteogenic activity, and is a promising bone tissue repair material.

Key words: Collagen special binding activity, Bone morphogenetic protein-2 mimetic peptide, Poly(lactic-co-glycolic acid), 3D printing scaffold, Osteogenic induction activity

中图分类号:

O631

TrendMD:

刘国民, 卢天成, 冀璇, 贾汶沅, 李亚龙, 赵一安, 罗云纲. 与胶原特异性结合的BMP2模拟肽/PLGA 3D打印复合支架的制备及成骨诱导活性. 高等学校化学学报, 2019, 40(7): 1552.

LIU Guomin, LU Tiancheng, JI Xuan, JIA Wenyuan, LI Yalong, ZHAO Yian, LUO Yungang. Preparation and Osteogenic Induction Activity of CBD-BMP2-MP/PLGA 3D Printed Composite Scaffolds^†. Chem. J. Chinese Universities, 2019, 40(7): 1552.

图/表 10

Table 1 Amino acid sequences of synthetic polypeptides

Number	Polypeptide	Peptide sequence/marker
1	BMP2 MP	RKIPKASSVPTELSAISMLYL
2	FITC-BMP2 MP	FITC-RKIPKASSVPTELSAISMLYL
3	CBD-BMP2 MP	WREPSFCALSGGGRKIPKASSVPTELSAISMLYL
4	FITC-CBD-BMP2 MP	FITC-WREPSFCALSGGGRKIPKASSVPTELSAISMLYL

Table 2 Real-time PCR primer

Gene	Primer sequence	Gene	Primer sequence
Runx2	5'-GACGTGCCCAGGCGTATTTC-3'(Forward)	OPN	5'-TCGGTACTGGTGTACCTGCT-3'(Reverse)
	5'-AAGTCTGGGGTCCGTCAAGG-3'(Reverse)	COL-I	5'-GAGCGGAGAGTACTGGATCG-3'(Forward)
ALP	5'-GCCCTCCAGATCCTGACCAA-3'(Forward)		5'-GCTTCTTTTCCTTGGGGTTC-3'(Reverse)
	5'-GCAGAGCCTGCTTGGCCTTA-3'(Reverse)	GAPDH	5'-AATGTGTCCGTCGTGGATCTG-3'(Forward)
OPN	5'-TCTCCTTGCGCCACAGAATG-3'(Forward)		5'-CAACCTGGTCCTCAGTGTAGC-3'(Reverse)

Fig.1 SEM images of PLGA/COL scaffolds(A—C), interpore structure of the PLGA/COL scaffolds(D—F) and pure PLGA scaffold(G—I)

Fig.2 Water droplets and contact angles on the surface of different scaffolds(A) PLGA; (B) PLGA/COL; (C) BMP2-MP@PLGA/COL; (D) CBD-BMP2-MP@PLGA/COL. P<0.05, n=4.

Fig.3 Mechanical properties of the PLGA(a), PLGA/COL(b), BMP2-MP@PLGA/COL(c) and CBD-BMP2-MP@PLGA/COL(d) tested at room temperature (A) Typical stress-strain curves; (B) compressive strength. n=3, P<0.05.

Fig.4 Collagen-binding ability of BMP2-MP and CBD-BMP2-MP detected by immunofluorescence (A) Immunofluorescence image; (B) total signal. Ex=488 nm, Em=546 nm, error bars represent standard deviation for n=3, * P<0.05.

Fig.5 Fluorescent staining observation of the MC3T3-E1 cells cultured on different composites for 3 d (A)PLGA; (B) PLGA/COL; (C) BMP2-MP@PLGA/COL; (D) CBD-BMP2-MP@PLGA/COL).

Fig.6 Proliferation of MC3T3-E1 cells in different porous scaffolds by CCK-8(* P<0.05, n=4) PLGA; PLGA/COL; BMP2-MP@PLGA/COL; CBD-BMP2-MP@PLGA/COL.

Fig.7 Alizarin Red staining of MC3T3-E1 cell cultured on PLGA(A, a), PLGA/COL(B, b), BMP2-MP@PLGA/COL(C, c), CBD-BMP2-MP@PLGA/COL)(D, d) at 14 d and their Ca2+ detection(E)(* P<0.05, n=4)

Fig.8 MC-3T3E1 cells grown on different scaffolds in the expression levels of COL-1(A), OPN(B), Runx2(C) and ALP(D) genes related to osteoblastic differentiation after 3 d of culture(* P<0.05, n=3)a. PLGA; b. PLGA/COL; c. BMP2 MP@PLGA/COL; d. CBD-BMP2 MP@PLGA/COL.

参考文献 25

[1]	Shen H., Hu X.X., Yang F., Bei J. Z., Wang S. G., Acta Biomaterialia, 2010, 6, 455—465
[2]	Zhang J., Li J., Jia G., Jiang Y., Liu Q., Yang X., RSC Advances, 2017, 7, 56732—56742
[3]	Toosi S., Naderi-Meshkin H., Kalalinia F., Peivandi M.T., HosseinKhani H., Bahrami A. R., Journal of Biomedical Materials Research Part A, 2016, 104, 2020—2028
[4]	Corcione C.E., Scalera F., Gervaso F., Montagna F., Sannino A., Maffezzoli A., J. Therm. Anal. Calorim., 2018, 134, 575—582
[5]	Gao T. L., Zhang N., Wang Z. L., Wang Y.,Liu Y., Ito Y., Macromol. Biosci., 2015, 15, 1070—1080
[6]	Yang X., Li Y. Y. He W., Huang Q. L., Zhang R. R., Feng Q. L., Journal of Biomedical Materials Research, Part A, 2018, 106, 2863—2870
[7]	Hwang K.S., Choi J. W., Kim J. H., Chung H. Y., Jin S., Shim J. H., Materials, 2017, 10(4), 421—434
[8]	Bae E.B., Park K. H., Shim J. H., Chung H. Y., Choi J. W., Lee J. J., Biomed. Res. Int., 2018, 10, 135—147
[9]	Kim B.R., Nguyen T. B. L., Min Y. K., Lee B. T., Tissue Eng. Part A, 2014, 20, 3279—3289
[10]	Shen H., Hu X.X., Yang F., Bei J. Z., Wang S. G., Biomaterials, 2011, 32, 3404—3412
[11]	Yan X., Chen B., Lin Y., Li Y. J., Xiao Z. F.,Hou X. L., Diabetes Res. Clin. Pr., 2010, 90, 66—72
[12]	Shao L., Haltiwanger R. S., Cell Mol. Life Sci., 2003, 60, 241—250
[13]	Zhang C., Miyatake H., Wang Y., Inaba T., Wang Y., Zhang P. B., Angew Chem. Int. Ed., 2016, 55, 11447—11451
[14]	Wang Y., Fu C., Wu Z. X., Chen L.,Chen X. S., Wei Y., Carbohyd. Polym., 2017, 174, 723—730
[15]	Tada S., Kitajima T., Ito Y., Int. J. Mol. Sci., 2012, 13, 6053—6072
[16]	Zhu W.G., Qiu Y., Sheng F., Yuan X. X., Xu L. L., Bao H. D., J. Mater. Sci-Mater M, 2018, 29(1), 2
[17]	Wang H.Y., Feng Y. K., An B., Zhang W. C., Sun M. L., Fang Z. C., J. Mater. Sci-Mater M, 2012, 23, 1499—1510
[18]	Walz J. A., Mace C. R., Anal. Chem., 2018, 90, 6572—6579
[19]	Won J.Y., Park C. Y., Bae J. H., Ahn G., Kim C., Lim D. H., Biomedical Materials, 2016, 11(5), 055013
[20]	Zhou J., Guo X.D., Zheng Q. X., Wu Y. C., Cui F. Z., Wu B., Colloid Surface B, 2017, 152, 124—132
[21]	Lai G. J., Shalumon K. T.,Chen S. H., Chen J. P., Carbohyd. Polym., 2014, 111, 288—297
[22]	Hughes-Fulford M., Li C. F., J. Orthop. Surg. Res., 2011, 6, 1186—1194
[23]	Cui H.T., Wang Y., Cui L. G., Zhang P. B., Wang X. H., Wei Y., Biomacromolecules, 2014, 15, 3146—3157
[24]	Wu J.P., Li L. L, Fu C., Yang F., Jiao Z. X., Shi X. C., Colloid Surface B, 2018, 169, 233—241
[25]	Li X. Y, Wang Y., Guo M., Wang Z. L., Shao N. N.,Zhang P. B., ACS Sustain Chem. Eng., 2018, 6, 2047—2054