Plasmid DNA-loaded asymmetrically porous membrane for guided bone regeneration

doi:10.1016/j.jmst.2020.03.015

Abstract

Abstract:

Although bone defects can be restored spontaneously, bone reconstruction with sufficient strength and volume continues to be a challenge in clinical practices. In recent years, the use of a variety of biomaterials with bioactivity has been attempted to compensate for this limitation. Herein, we fabricated a pDNA (encoding for BMP-2)-loaded asymmetrically porous polycaprolactone (PCL)/Pluronic F127 membrane as a bioactive guided bone regeneration (GBR) membrane, using a modified immersion-precipitation method. It was observed that the GBR membrane allows continuous release of pDNA for more than 20 weeks. The pDNA was sufficiently transfected into human bone marrow stem cells (hBMSCs) without significant cytotoxicity and the gene-transfected cells showed prolonged synthesis of BMP-2. From in vitro osteogenic differentiation and in vivo animal studies, the effective induction of osteogenic differentiation of hBMSCs and enhanced bone regeneration by the pDNA-loaded asymmetrically porous PCL/Pluronic F127 membrane was observed, suggesting that the pDNA-loaded membrane as a bioactive GBR membrane can be an alternative therapeutic technique for effective bone regeneration.

Key words: Plasmid DNA (pDNA), Guided bone regeneration (GBR), Bone morphogenetic protein-2 (BMP-2), Polycaprolactone (PCL), Gene delivery

Se Heang Oh, June-Ho Byun, So Young Chun, Young-Joo Jang, Jin Ho Lee. Plasmid DNA-loaded asymmetrically porous membrane for guided bone regeneration[J]. J. Mater. Sci. Technol., 2021, 63: 161-171.

Figures/Tables 9

Fig. 1. Photographs showing the animal study (using miniature pig) procedure for implantation of hBMSCs- and/or pDNA-loaded asymmetrically porous PCL/Pluronic F127 (5 wt%) membrane to compare bone regeneration behavior (dotted circles, bony defects).

Fig. 2. (A) Transfection efficiency and (B) cell viability of hBMSCs by pDNA complexes (pDNA/PEI-PEG) with different N/P ratios (n = 3; *p < 0.05).

Fig. 3. Characterization of the pDNA-loaded asymmetrically porous membranes. (A) SEM images showing the morphology of the pDNA-loaded PCL/Pluronic F127 (5 wt%) membrane, (B) Tensile strength of the pDNA-loaded PCL/Pluronic F127 membranes with different Pluronic F127 compositions, and (C) Release patterns of pDNA from the hydrophobic PCL and hydrophilized PCL/Pluronic F127 (5 wt%) membranes (n = 3).

Fig. 4. (A) Transfection efficiency and (B) cell viability of hBMSCs by pDNA complexes (N/P ratio 16) released from hydrophobic PCL and hydrophilized PCL/Pluronic F127 (5 wt%) membranes (green color, transfected hBMSCs, x 200; n = 3; *p < 0.05).

Fig. 5. ELISA result on BMP-2 synthesis from hBMSCs transfected by pDNA released from the hydrophobic PCL and hydrophilized PCL/Pluronic F127 (5 wt%) membranes (n = 3).

Fig. 6. In vitro osteogenic differentiation behavior of hBMSCs incubated with pDNA-loaded hydrophobic PCL and hydrophilized PCL/Pluronic F127 (5 wt%) membranes. (A) Calcium content, (B) quantitative real-time PCR (osteocalcin and osteopontin), (C) western blotting (osteopontin; 4 weeks of cell culture), and (D) immunocytochemical analysis [osteocalcin (red color), cell nuclei (blue); 4 weeks of cell culture] (n = 3; *p < 0.05).

Fig. 7. In vivo bone regeneration behavior among the different GBR membrane groups (SD rat, calvarial defect). (A) Micro-CT and (B) bone fraction in defect area (dotted circles, bony defects; n = 3; *p < 0.05 compared with hBMSCs/pDNA-membrane group, #p < 0.05 compared with pDNA-membrane group, &p < 0.05 compared with pDNA-membrane group, $p < 0.05 compared with Membrane group, and @p < 0.05 compared with Blank group).

Fig. 8. In vivo bone regeneration behaviors among the different GBR membrane groups (miniature pig, mandibular bone defect). (A) Plain radiographic images, (B) CT images, (C) three-dimensional CT reconstructions images, and (D) histological sections (H&E staining; after 12 weeks) from the reconstructed bone region (dotted circle, bony defect; dotted line, border between host and regenerated bones; arrow, Harversian system).

Table 1 HU values of reconstructed bone defects after the implantation of different GBR membrane groups (miniature pig, mandibular bone defect).

Groups		Weeks
Groups		0	4	8	12
Animal 1
hBMSCs/pDNA-membrane	Mean	120.80	245.91	674.63	1110.37
hBMSCs/pDNA-membrane	SD	297.03	320.79	558.02	534.38
pDNA-membrane	Mean	-286.23	242.71	391.88	624.72
pDNA-membrane	SD	652.02	260.65	427.27	522.94
hBMSCs-membrane	Mean	-120.95	187.13	592.24	715.89
hBMSCs-membrane	SD	393.14	264.18	469.50	517.63
Membrane	Mean	-494.52	269.00	406.03	415.33
Membrane	SD	395.42	284.40	323.45	387.85
Animal 2
hBMSCs/pDNA-membrane	Mean	-267.28	248.23	705.07	1024.37
hBMSCs/pDNA-membrane	SD	478.79	303.67	477.51	454.87
pDNA-membrane	Mean	-118.70	260.34	496.82	776.87
pDNA-membrane	SD	758.13	325.43	482.21	468.64
hBMSCs-membrane	Mean	-275.92	275.85	421.15	665.21
hBMSCs-membrane	SD	367.63	286.34	319.31	378.71
Membrane	Mean	-337.84	221.87	427.79	520.63
Membrane	SD	470.32	254.07	350.23	410.52

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