含骨脱细胞基质电纺纤维的成骨性能

doi:10.7503/cjcu20190524

摘要/Abstract

摘要：

通过脱细胞技术制备了猪骨脱细胞基质(DBM), 用胃蛋白酶消化DBM使其变为可溶形式, 采用静电纺丝技术制备了含有DBM的左旋聚乳酸(PLLA)电纺纤维(PLLA/DBM), 并对PLLA/DBM的形貌、亲水性、细胞相容性、成骨性能和体外矿化能力进行评价. 研究结果表明, 脱细胞处理能够有效去除骨组织中的细胞成分, 使DNA含量显著下降. DBM经胃蛋白酶处理后溶于六氟异丙醇(HFIP), 可进行静电纺丝, 制备的PLLA/DBM[m(PLLA)∶m(DBM)=10∶0, 9∶1, 7∶3, 5∶5]电纺纤维具有良好的亲水性, 且无细胞毒性, 对骨髓间充质干细胞的黏附及成骨分化有明显的诱导促进作用, 体外生物矿化效果优良.

关键词: 细胞外基质, 骨脱细胞基质, 静电纺丝, 骨组织工程

Abstract:

Porcine decellularized bone matrix(DBM) was firstly prepared, and then solubilized via pepsin digestion. Solubilized DBM and poly(L-lactic acid)(PLLA) were subsequently processed into PLLA/DBM electrospun nanofibers through eletrospinning technology. The morphology, wettability and mineralization ability of PLLA/DBM electrospun nanofibers were examined, and cytocompatibility and osteogenic differentiation capacity were evaluated by culturing bone marrow mesenchymal cells(BMSCs) on PLLA/DBM electrospun nanofibers. The results showed that cellular components in cancellous bone could be effectively removed after decellularization, as evidenced by the significant decrease in DNA content. The solubilized DBM could be dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol(HFIP) for electrospinning of the PLLA/DBM composite nanofibers formulated at different mass ratios(10∶0, 9∶1, 7∶3 and 5∶5). The obtained PLLA/DBM electrospun nanofibers were demonstrated to be hydrophilic and cytocompatible, and significantly promoted the attachment and osteogenic differentiation of the BMSCs. Furthermore, the presence of DBM facilitated mineral deposition on the nanofiber surface in simulated body fluid(SBF), which suggests its great potential in promoting bonding with the natural bone tissue.

Key words: Extracellular matrix, Decellularized bone matrix, Electrospinning, Bone tissue engineering

中图分类号:

O636

TrendMD:

秦春萍, 王先流, 唐寒, 易兵成, 刘畅, 张彦中. 含骨脱细胞基质电纺纤维的成骨性能. 高等学校化学学报, 2020, 41(4): 780.

QIN Chunping, WANG Xianliu, TANG Han, YI Bingcheng, LIU Chang, ZHANG Yanzhong. Osteogenesis-promoting Effects of the Electrospun Nanofibers Containing Decellularized Bone Matrix ^†. Chem. J. Chinese Universities, 2020, 41(4): 780.

图/表 9

Fig.1 Optical(A1, A2), HE staining(B1, B2) and Masson trichrome staining(C1, C2) images of demineralized bone matrix(A1—C1) and decellularized bone matrix(A2—C2)

Fig.2 DNA content comparison between decellularized bone matrix and the native bone(**P< 0.01)

Fig.3 SEM images(A—D) and the corresponding diameter distributions(E—H) of PLLA/DBM-10/0(A, E), PLLA/DBM-9/1(B, F), PLLA/DBM-7/3(C, G) and PLLA/DBM-5/5(D, H) electrospun nanofibers

Fig.4 FTIR spectra(A) and water contact angles(B) of PLLA/DBM electrospun nanofibers (A) a. DBM; b. PLLA/DBM-10/0; c. PLLA/DBM-9/1; d. PLLA/DBM-7/3; e. PLLA/DBM-5/5. (B) a. PLLA/DBM-10/0; b. PLLA/DBM-9/1; c. PLLA/DBM-7/3; d. PLLA/DBM-5/5.

Fig.5 Cell proliferation of BMSCs on PLLA/DBM electrospun nanofibersa. PLLA/DBM-10/0; b. PLLA/DBM-9/1; c. PLLA/DBM-7/3; d. PLLA/DBM-5/5; e. TCP. *P< 0.05, **P< 0.01.

Fig.6 Cell morphologies of BMSCs cultured on PLLA/DBM-10/0(A, C) and PLLA/DBM-7/3(B, D) electrospun nanofibers after 2 h(A, B) and 24 h(C, D)

Fig.7 ALP staining(A, B), quantified ALP activity(C, **P<0.01), ARS staining(D, E) and ARS quantification(F, **P<0.01) of BMSCs cultured on PLLA/DBM electrospun nanofibers(A), (D) PLLA/DBM-10/0; (B), (E) PLLA/DBM-7/3.

Fig.8 SEM images(A—D) and XRD patterns(E) of PLLA/DBM electrospun nanofibers after being incubated in SBF(A) PLLA/DBM-10/0, 7 d; (B) PLLA/DBM-7/3, 7 d; (C) PLLA/DBM-10/0, 14 d; (D) PLLA/DBM-7/3, 14 d. (E) a. PLLA/DBM-7/3; b. PLLA/DBM-7/3 after mineralization for 14 d; c. PLLA/DBM-10/0; d. PLLA/DBM-10/0 after mineralization for 14 d.

Table 1 Chemical composition of PLLA/DBM electrospun nanofibers after being incubated in SBF by EDS analysis

Sample	Atomic fraction(%)
Sample	C	O	P	Ca	Mg
PLLA/DBM-10/0	67.69	32.27	0.01	0.03	0
PLLA/DBM-7/3	17.66	52.69	10.43	18.60	0.62

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