Preparation and Osteogenic Induction Activity of CBD-BMP2-MP/PLGA 3D Printed Composite Scaffolds†

doi:10.7503/cjcu20190145

Abstract

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

CLC Number:

O631

TrendMD:

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^†[J]. Chem. J. Chinese Universities, 2019, 40(7): 1552.

Figures/Tables 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.

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