The Combination of Hydrogels with 3D Fibrous Scaffolds Based on Electrospinning and Meltblown Technology

Abstract

This study presents the advantages of combining three-dimensional biodegradable scaffolds with the injection bioprinting of hydrogels. This combination takes advantage of the synergic effect of the properties of the various components, namely the very favorable mechanical and structural properties of fiber scaffolds fabricated from polycaprolactone and the targeted injection of a hydrogel cell suspension with a high degree of hydrophilicity. These properties exert a very positive impact in terms of promoting inner cell proliferation and the ability to create compact tissue. The scaffolds were composed of a mixture of microfibers produced via meltblown technology that ensured both an optimal three-dimensional porous structure and sufficient mechanical properties, and electrospun nanofibers that allowed for good cell adhesion. The scaffolds were suitable for combination with injection bioprinting thanks to their mechanical properties, i.e., only one nanofibrous scaffold became deformed during the injection process. A computer numerical-control manipulator featuring a heated printhead that allowed for the exact dosing of the hydrogel cell suspension into the scaffolds was used for the injection bioprinting. The hyaluronan hydrogel created a favorable hydrophilic ambiance following the filling of the fiber structure. Preliminary in vitro testing proved the high potential of this combination with respect to the field of bone tissue engineering. The ideal structural and mechanical properties of the tested material allowed osteoblasts to proliferate into the inner structure of the sample. Further, the tests demonstrated the significant contribution of printed hydrogel-cell suspension to the cell proliferation rate. Thus, the study led to the identification of a suitable hydrogel for osteoblasts.

Keywords: bioprinting; electrospinning; hydrogels; meltblown; nanofibers; scaffold.

Figure 1

Scheme of a combination of meltblown and electrospinning technology for the production of a 3D scaffold: 1—extruder, 2—high voltage power supply, 3—multi needle spinner, 4—drum collector.

Figure 2

Manufactured 3D scaffolds combining micro- and nanofibers. (A–C)—scaffold morphology observed with SEM. (D)—the image of scaffolds in the form of discs. Table—structural parameters of the material [17].

Figure 3

Three-dimensional scaffolds with a pressed trap hole. (A)—image of scaffolds with a pressed hole. (B)—SEM image of a pressed hole. (C)—SEM detail image of the hole wall.

Figure 4

Overall diagram of the bioprinter (A); printhead diagram (B); printhead detail (C). (A) 1—printhead, 2—frame, 3—well plate, 4—x and z axis drive, 5—printing medium, 6—case, 7—heating spiral, 8—needle, 9—fiber scaffold sample, 10—extruded medium inside the scaffold.

Figure 5

Images from fluorescence microscopy of bone osteoblasts on scaffolds on the 1st, 4th and 7th test days—propidium iodide staining. The images consist of shots taken by microscope auto-focus in z-axis, 1 µm step; the scale is 1 mm. SC—scaffold with conventionally seeded cells, HG—scaffolds with printed hydrogel.

Figure 6

Cell metabolic activity described by the MTT Assay—comparison of surface bioprinting and conventional cell seeding (mean value ± standard deviation, n = 4). Statistically significant difference in the significance level: *** p < 0.0006; **** p < 0.0001. SC—scaffolds with conventionally seeded cells, HG—scaffolds with a printed hydrogel bioink.

Figure 7

Images of fluorescence microscopy of bone osteoblasts on scaffolds on the 1st, 4th and 7th test days comparing the effect of the internal printing of various bioinks on the viability of human osteoblasts—propidium iodide staining. The images consist of shots taken by microscope auto-focus in z-axis, 1 µm step; the scale is 500 µm. The top section shows surface cell proliferation—OUTSIDE. The bottom part shows internal cell proliferation (3 mm under the surface)—INSIDE. C—Hydrogel with incorporated collagen, HP—Hydrogel with incorporated heparin, PEG—polyethylene glycol photosensitive hydrogel, SC—scaffold with conventionally seeded cells.

Figure 8

Cell metabolic activity described by the MTT Assay—comparison of the effect of internal bioprinting of different print media on the viability of human osteoblasts (mean value ± standard deviation, n = 4). Statistically significant difference in the significance level: * p < 0.04; ** p < 0.002; **** p < 0.0001 (ANOVA, post-hoc Tukey). C—Hydrogel with incorporated collagen, HP—Hydrogel with incorporated heparin, PEG—polyethylene glycol photosensitive hydrogel. SC—scaffold with conventionally seeded cells.

Figure 9

Fluorescence images of live and dead bone osteoblasts on scaffolds of the 1st, 4th and 7th test days comparing the effect of the internal printing of selected bioink and scaffold without bioink on the viability of human osteoblasts—live cells (green: Calcein AM) and dead cells (red: EthD-I) staining. The scale is 100 µm. The top section shows surface cell proliferation—OUTSIDE. The bottom part shows internal cell proliferation (3 mm under the surface)—INSIDE. C—Hydrogel with incorporated collagen, SC—scaffold with conventionally seeded cells.

Figure 10

Cell proliferation rate described by cell number on 1 mm²—comparison of the effect of internal bioprinting of selected bioink and scaffold without bioink on the viability of human osteoblasts (mean value ± standard deviation, n = 10). Statistically significant difference in the significance level: ** p < 0.0046 (ANOVA, post-hoc Tuckey). The top section shows the number of cells per mm² on the surface. The bottom part shows the number of cells per mm² into the structure. C—Hydrogel with incorporated collagen, SC—scaffold with conventionally seeded cells, Dead—Number of dead cells.

Figure 11

Computed tomography images visualizing the distribution of hydrogel within a scaffold structure. (A)-side view of the sample; X-ray scan. (B)-3D sample screening.

Figure 12

Measurement of cyclic deformation. (A) The testing of reversible cyclic deformations of PCL scaffolds at sample compression values 30, 60, and 90%. (B) The deformation curves of cyclic deformations testing at sample compression values of 30, 60, and 90%.