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. 2019 Mar 26:14:2127-2144.
doi: 10.2147/IJN.S192699. eCollection 2019.

Electrospun polycaprolactone/collagen nanofibers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/ N- hydroxysuccinimide and genipin facilitate endothelial cell regeneration and may be a promising candidate for vascular scaffolds

Dian Chen  1 , Tonghe Zhu  2   3 , Wei Fu  4 , Haibo Zhang  1
Affiliations

Electrospun polycaprolactone/collagen nanofibers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/ N- hydroxysuccinimide and genipin facilitate endothelial cell regeneration and may be a promising candidate for vascular scaffolds

Dian Chen et al. Int J Nanomedicine. .

Abstract

Purpose: A promising vascular scaffold must possess satisfying mechanical properties, great hemocompatibility, and favorable tissue regeneration. Combining natural with synthetic materials is a popular method of creating/enhancing such scaffolds. However, the effect of additional modification on the materials requires further exploration.

Materials and methods: We selected polycaprolactone (PCL), which has excellent mechanical properties and biocompatibility and can be combined with collagen. Electrospun fibers created using a PCL/collagen solution were used to fashion mixed nanofibers, while separate syringes of PCL and collagen were used to create separated nanofibers, resulting in different pore sizes. Mixed and separated nanofibers were cross-linked with glutaraldehyde (GA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and genipin; hence, we named them as mixed GA, mixed EDC (ME), mixed genipin (MG), separated GA, separated EDC (SE), and separated genipin (SG).

Results: Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction showed that cross-linking did not affect the main functional groups of fibers in all groups. ME, MG, SE, and SG met the requisite mechanical properties, and they also resisted collagenase degradation. In hemocompatibility assays, only ME and MG demonstrated ideal safety. Furthermore, ME and MG presented the greatest cytocompatibility. For vascular scaffolds, rapid endothelialization helps to prevent thrombosis. According to human umbilical vein endothelial cell migration on different nanofibers, ME and MG are also successful in promoting cell migration.

Conclusion: ME and MG may be promising candidates for vascular tissue engineering. The study suggests that collagen cross-linked by EDC/N-hydroxysuccinimide or genipin facilitates endothelial cell regeneration, which could be of great benefit in tissue engineering of vascular scaffolds.

Keywords: glutaraldehyde; hemocompatibility; mechanical test; migration assay; subcutaneous implantation; tissue engineering.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Schematic of the cross-linking reactions of collagen with GA (A), EDC/NHS (B), and genipin (C). Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; NHS, N-hydroxysuccinimide.
Figure 2
Figure 2
SEM images of different cross-linked membranes under mixed 10% (w/v) solution of PCL/collagen (A–D) and separated 10% (w/v) PCL and 10% (w/v) collagen solution (E–H). (I, J) Fiber diameter distribution of MF and SF, respectively. Notes: Magnification, 10,000× times. Scale bar, 8 µm. Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; NHS, N-hydroxysuccinimide; PCL, polycaprolactone; SEM, scanning electron microscopy; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 3
Figure 3
Water contact angles of different cross-linked membranes at desired time points (0, 20, 50, and 90 seconds). Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; ME, mixed EDC; MG, mixed genipin; MGA, mixed GA; SE, separated EDC; SG, separated genipin; SGA, separated GA; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 4
Figure 4
FTIR spectra of fresh and different cross-linked membranes. Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; FTIR, Fourier transform infrared; GA, glutaraldehyde; ME, mixed EDC; MG, mixed genipin; MGA, mixed GA; SE, separated EDC; SG, separated genipin; SGA, separated GA; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 5
Figure 5
XRD pattern of fresh and different cross-linked membranes. Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; ME, mixed EDC; MG, mixed genipin; MGA, mixed GA; PCL, polycaprolactone; SE, separated EDC; SG, separated genipin; SGA, separated GA; XRD, X-ray diffraction; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 6
Figure 6
Mechanical assays for different cross-linked membranes. Notes: (A) Curve of stress on tensile process. (B) UTS. (C) Elongation percentage on the break of tensile process. *A statistically significant difference for the mean value compared with all other membranes. #Denotes a statistically significant difference for the mean value compared with other membranes except the group with the same # symbol (P<0.05, n=4). Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; ME, mixed EDC; MG, mixed genipin; MGA, mixed GA; SE, separated EDC; SG, separated genipin; SGA, separated GA; UTS, ultimate tensile stress; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 7
Figure 7
Degradation test for different cross-linked membranes. Notes: (A) SEM images of different cross-linked membranes after soaked in degradation solution on the 15th day. Magnification, 300× times; scale bar, 200 µm. Magnification, 1,500× times; scale bar, 50 µm. (B) Weight remaining in different cross-linked membranes after incubating at 37°C in PBS with collagenase type I. *A statistically significant difference (P<0.05, n=4) in the mean value compared with the last time point. Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; ME, mixed EDC; MG, mixed genipin; MGA, mixed GA; SE, separated EDC; SEM, scanning electron microscopy; SG, separated genipin; SGA, separated GA; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 8
Figure 8
(A) CCK-8 assay of HUVEC proliferation adhered on the coverslips and different cross-linked membranes at desired time points (1, 4, and 7 days after operation). The sequence of letters a–d represents the size of the mean value (a>b>c>d). The same letter indicates no statistically significant difference (P<0.05, n=4). (B) SEM observations of HUVECs grown on the different cross-linked membranes at 1 and 4 days after culture. Magnification, 250×. Scale bar, 300 µm. Abbreviations: CCK-8, Cell Counting Kit-8; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; HUVECs, human umbilical vein endothelial cells; ME, mixed EDC; MG, mixed genipin; MGA, mixed GA; NHS, N-hydroxysuccinimide; SE, separated EDC; SEM, scanning electron microscopy; SG, separated genipin; SGA, separated GA; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 9
Figure 9
Hemolytic assay of different cross-linked membranes. Notes: *A statistically significant difference in the mean value compared with all other membranes. #Denotes a statistically significant difference in the mean value compared with water and PBS (P<0.05, n=8). Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; ME, mixed EDC; MG, mixed genipin; MGA, mixed GA; SE, separated EDC; SG, separated genipin; SGA, separated GA; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 10
Figure 10
Platelets deposited on different membranes, quantified by LDH activity. Notes: The sequence of letters a–c represents the size of the mean value (a>b>c). The same letter indicates no statistically significant difference (P<0.05, n=4). Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; LDH, lactate dehydrogenase; ME, mixed EDC; MG, mixed genipin; MGA, mixed GA; SE, separated EDC; SG, separated genipin; SGA, separated GA; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 11
Figure 11
Cross-sections within immunohistochemical analysis of different cross-linked membranes after subcutaneous implantation. Notes: The scale bar of first and third line, 50 µm. The scale bar of second and last line, 100 µm. Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; ME, mixed EDC; MG, mixed genipin; MGA, mixed GA; SE, separated EDC; SG, separated genipin; SGA, separated GA; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 12
Figure 12
Migration of HUVECs on the coverslips and different cross-linked membranes with DAPI staining at desired time points (0 hour, 1 day, 4 days, 7 days). Scale bar, 4,000 µm. Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; HUVECs, human umbilical vein endothelial cells; ME, mixed EDC; MG, mixed genipin; MGA, mixed GA; SE, separated EDC; SG, separated genipin; SGA, separated GA; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 13
Figure 13
(A) Migration area of HUVECs on different membranes at designated time points (0 hour, 1 day, 4 days, and 7 days). (B) HUVECs migrated outward over 7 days on different membranes. *Denotes a statistically significant difference in the mean value compared with other membranes (P<0.05, n=3). Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; HUVECs, human umbilical vein endothelial cells; ME, mixed EDC; MG, mixed genipin; MGA, mixed GA; SE, separated EDC; SG, separated genipin; SGA, separated GA; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 14
Figure 14
H&E staining images of different cross-linked membranes implanted subcutaneously at 3, 7, 10, 20, and 30 days. Notes: Depth of the cells infiltrating into the membranes as a percentage of the depth of the test membrane. The dotted line is the boundary between the host tissue and the material. A1–A4, B1–B4, C1–C4, D1–D4 and E1–E4 are H&E staining images. F is the depth of the cells infiltrated into test samples. Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; NHS, N-hydroxysuccinimide; SE, separated EDC; SG, separated genipin; SGA, separated GA; MF, fresh mixed membranes; SF, fresh separated membranes.
Figure 14
Figure 14
H&E staining images of different cross-linked membranes implanted subcutaneously at 3, 7, 10, 20, and 30 days. Notes: Depth of the cells infiltrating into the membranes as a percentage of the depth of the test membrane. The dotted line is the boundary between the host tissue and the material. A1–A4, B1–B4, C1–C4, D1–D4 and E1–E4 are H&E staining images. F is the depth of the cells infiltrated into test samples. Abbreviations: EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GA, glutaraldehyde; NHS, N-hydroxysuccinimide; SE, separated EDC; SG, separated genipin; SGA, separated GA; MF, fresh mixed membranes; SF, fresh separated membranes.
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