3D printed high-resolution scaffold with hydrogel microfibers for providing excellent biocompatibility

Materials

In this study, the composite scaffolds consist of MEW scaffolds and the HFM. The MEW scaffolds fabricated by polycaprolactone (PCL, CAPA6800, Perstorp Ltd., UK), which the molecular weight of PCL was 80,000 g/mol and the melting temperature was 60 °C. The HFM was fabricated by Gelatin methacryloyl (GelMA, EFL-GM-500, Yongqinquan Intelligent Equipment Co., Ltd., Suzhou, China) and polyacetylene (PEO, Sigma-Aldrich, America, the relative molecular weight of 1 million). The spinning solutions were composed of 10% (w/v) GelMA, 1.5% (w/v) PEO, 0.5%(w/v)lithium phenyl-2,4,6-trimethyl-benzoyl phosphinate(Photoinitiator 2959, Suzhou Intelligent Manufacturing Research Institute, China), and 75% alcohol, placed at 37 °C and stirring overnight. The state of the mixed material is shown in Figure S1 and Figure S2.

Rheological characterization

At room temperature, a rheometer (DISCOVERY HR-2) was used to perform rheological-shear thinning tests on GelMA solutions and GelMA/PEO solutions to simulate the effect of mechanical extrusion speed on spinning during printing.

Devices

Fabrication strategy of composite scaffolds

As shown in Figure 2, the composite scaffolds consisted of the MEW scaffolds and the GElMA/PEO HFM. The MEW scaffolds were fabricated by melt electrowriting (Figure 2(a)). The PCL was loaded into a metal cylinder, and the parameters were set as follows: nozzle temperature, 95 °C; syringe temperature, 80 °C; air pressure, 9KPa; and voltage, 4.6 kV; collector speed, 1200 mm/min; and collector distance, 2.5 mm. The MEW scaffolds were then placed in the electrospinning receiving plane after fabrication and GelMA HFM by electrospinning about 2 h (Figure 2(b)). The spinning parameters were included: voltage, 11 kV, flow rate, 0.5 ul/min, and collector distance, 40 mm. After electrospinning, the composite scaffold was placed in a saturated ammonium sulfate solution and irradiated with a 250–365 nm ultraviolet light system for 1 minute to form a stable scaffold. The composite scaffolds were ultimately moved to the PBS solution and stored at room temperature.

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Figure 2.

Design and fabricate of the composite scaffold. (a) Fabricate of MEW scaffold by melting electrowriting; (b) electrospinning GelMA fibers membrane on the MEW scaffold; (c) The composite scaffold was crosslinked in an ammonium sulfate solution.

Morphology analysis of composite scaffold

The scanning electron microscopy (JSM-IT100, Japan) was used to the morphology of MEW scaffolds, composite scaffolds before cross-linking, and scaffolds after cross-linking. Before observation, all samples were sprayed with gold for 2 min.

Cell culture

To research the behavior of cells on MEW scaffolds, coated scaffolds, and composite scaffolds, respectively. HUVES(Suzhou Intelligent Manufacturing Research Institute, Suzhou, China) were cultured in DMEM(Tomp Biotechnology Co., LTD., China) with 10% fetal bovine serum (100 units/ml, Tomp Biotechnology Co., LTD., China)and 1% penicillin/streptomycin (100 µg/ml, Tomp Biotechnology Co., LTD., China). Before seeding the cells, the scaffolds of different groups were sterilized in 75% alcohol for two hours. Subsequently, the sterilized scaffolds were transferred to 24-well plates with a density of 1 × 10⁵ cells/scaffold on each scaffold's surface. Finally, the scaffolds were placed in an incubator at 37 °C and 5% CO₂ for incubation.

Cell proliferation

They were using Cell Counting kit-8 (CCK-8, Dojindo) to analyze cell proliferation. Different groups scaffolds at day1,4 and 7 were transferred to another 24-well plate and washed three times with PBS solutions(Tomp Biotechnology Co., LTD., China). Mix 50 µl CCK-8 and 450 µl DMEM medium and add to each well and incubate for 3 h. Subsequently, the solutions were aspirated and added to a 96-well plate, measure the absorbance at 450 nm and record it as the OD value. Each sample was tested 5 times. The MEW scaffold's value was recorded as OD₁, composite scaffold recorded as OD₂, and coated scaffold recorded as OD₃.

Cell viability

To analyze the viability of HUVES on different groups of scaffolds, the scaffolds were stained with LIVE/DEAD reagents (KeyGEN BioTECH Co., Ltd., Nanjing, China). The green living cells and red dead cells with a fluorescent confocal fluorescence microscope (OLYMPUS FV3000).

Cell morphology

The cells' morphology was analyzed by double staining the cells with DAPI (10 µg/ml; Solarbio, Beijing, China) and TRITC phalloidin (0.1 µM, YEASEN BioTECH Co., Ltd., Shanghai, China). Because DAPI can combine with the nucleus to produce blue fluorescence, phalloidin can bind to the F-actin of cells, and red rhodamine-labeled phalloidin can show the morphology of the cells. The stained scaffolds were observed under a fluorescent confocal microscope, and the cell images were processed with Image-J software.

Statistical analysis

All data in the experiment were expressed in the form of mean ± standard deviation (SD), and statistical analysis was performed using analysis of variance (ANOVA). A single asterisk(*) indicates 0.01<P < 0.05, and there are statistical differences between the groups. Double asterisks (**) indicate 0.001<P < 0.01, there are statistically significant differences between the groups. Three asterisks (***) indicate p < 0.001, and there are incredibly significant statistical differences between the groups. The NS indicates no statistically significant difference between the groups (p > 0.05).

Manufacturing process analysis of composite scaffolds

As a biological material, GelMA possesses excellent biocompatibility, biodegradation, and low antigenicity, similar to the ECM. ²⁷ However, the printability of GelMA is low, and application in biomedicine is limited, severely, due to low viscosity. ²⁹ It is challenging to fabricate GelMA fibers by electrospinning directly. To solve this dilemma, our study proposed strategy to addition PEO as a thickener in the GelMA solution to enhance the viscosity and improve the printability of GelMA. ³⁰ However, in fabricating GelMA HFM, the solution's viscosity plays a crucial part in the morphology and size of the fiber. Studies have shown that the range of solution viscosity is 0.1∼2 Pa·s and suitable for fibers' fabrication. ³¹ As shown in Figure 3(a), the rheology analysis of GelMA and PEO of different concentrations. The results show that the initial viscosity of 10% GelMA is lower than 0.1 Pa·s, while the initial viscosity of 10% GelMA + 2.5% PEO is as high as 4.29 Pa·s. These two ratios of mixed materials are challenging to make fibers because the solution's surface tension is the dominant factor in the case of low viscosity. It is difficult for the jet to form continuous fibers, and only droplets or microbead structures can be formed; when the viscosity is higher than one After the critical value, because the degree of entanglement between the molecular chains increases, the tension of the solution relaxes for a longer time, and it is difficult for the solution to eject from the spinneret to form a jet. ³¹ The rheological analysis showed that the initial viscosity of 10% GelMA + 0.5% PEO was 0.16 Pa·s, and the initial viscosity of 10% GelMA + 1.5% PEO was 1.75 Pa·s. Theoretically, these two groups of mixed materials' viscosity are suitable for fabricating fibers, but through spinning experiment tests, the fibers made from the mixed materials of 10% GelMA + 0.5% PEO show unevenness, and the fibers appear into droplets or microbeads. (Figure 3(b)(I)), because with the increase of the shear rate, the viscosity of the solution gradually decreases and is lower than 0.1 Pa·s, which makes the material of this concentration unsuitable for fabricating uniform fibers. Figure 3(a) shows that the 10%GelMA + 1.5%PEO mixed material's viscosity can be stabilized in the range of 0.1∼2 Pa·s. Therefore, under the electric field force's stretching action, it can be quickly continuously extruded from the nozzle to produce stable and uniform fibers (Figure 3(b)(II)).

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Figure 3.

Printability study of GelMA and the cross-linking strategy of the composite scaffold. (a) Rheological properties of different concentrations of GelMA and PEO mixed solutions; (b) (I) electrospinning of 10%GelMA + 0.5%PEO, (II) electrospinning of10%GelMA + 1.5%PEO; (c) the effect of fabricating parameters on the diameter of the fibers in electrospinning; (d) (I) The fiber diameter at 1.5 µl/min and 7 kV, (II) the fiber diameter at 0.5 µl/min and 11 kV.

The fiber morphology is affected by the factors, such as the ink properties, print parameters, and environmental parameters in electrospinning. ³² Fiber diameter is one of the most critical evaluation parameters. In our current research, the printability of GelMA/PEO was analyzed by adjusting the flow rate and voltage on fiber diameter time while the other parameters remained constant. It is observed that the diameter of fiber increases with the acceleration of flow rate, but decrease with the increase of voltage. The fiber diameter is 16.96 ± 1.28 µm when the flow rate is 1.5 µl/min (Figure 3 (d)(I)) because the solution's flow rate will vary with the flow rate. The low flow rate is conducive to the solvent volatilization and the fiber dissolution. It is accessible to formed beads; due to the high flow rate of solvent volatilization slow, the stretch phenomenon is not sufficient, increasing the fiber. ³³ Moreover, voltage is another significant parameter in electrospinning. The electric field force will overcome the surface tension of the solution when the critical voltage is reached, and the solution will be formed a jet ejected from the Taylor cone to form a jet, which will form fibers under the further stretching of the electric field force. ³⁴ The jet's charge density will increase with the promotion of voltage, which enhances the stretching effect of the jet and speeds up the solvent volatilization. Meanwhile, the Coulombic Repulsion effect will increase in the jet, which is beneficial to the fiber's refinement and the reduction of the fiber's diameter. As shown in Figure 3(d)(III), the fiber diameter is 2.94 ± 0.46 µm when the voltage reaches11 kV. The water in the fluid evaporates rapidly during the electrospinning, and the manufacturing lasts a long time so that the fiber fabricated is dry. Nevertheless, the cross-linking of GelMA HFM needs to be carried out in a liquid environment. How to solve this issue becomes a critical factor for the fabrication of composite scaffolds. If the fabricated GelMA HFM were cross-linked in an aqueous solution, it would dissolve rapidly because GelMA HFM has a high solubility in water and a smaller diameter. As GelMA was the product of modified gelatin, it has the essential characteristics of the protein and can take advantage of low protein solubility characteristics in the high concentration of metal salt. ³⁵ In this study, the composite scaffold was placed in a saturated ammonium sulfate solution, constructing a liquid cross-linking environment with low solubility of GelMA HFM and fabrication of composite scaffolds, successfully.

The microstructure of the different groups of scaffolds was observed by SEM in Figure 4, which exhibits the feasibility of manufacturing composite scaffolds and cross-linking strategy in a saturated ammonium sulfate solution. As shown in Figure 4(a), the MEW scaffolds fabricated by adjusting printing parameters and precise path control show the excellent structure and high porosity, and the diameter of the fibers of the scaffold is about 14.25 ± 3.86 µm, and the pore size is 150 µm. In Figure 4(b) shows the composite scaffold before cross-linking. The fiber's diameter is about 2.94 ± 0.46 µm of GelMA HFM. Figure 4(c) shows the composite scaffolds after cross-linking. It can be observed from the SEM that the GelMA HFM covers the surface of the MEW scaffolds uniformly, which significantly reduces the pore size of the scaffolds' surface and maintains the structure of the MEW scaffolds, providing an appropriate environment for cell adhesion. Figure 4(c) shows the composite scaffold after cross-linking in the ammonium sulfate solution. Compared with before the cross-linking, the composite scaffolds' morphology and structure remained almost unchanged after cross-linking, and the GelMA HFM still covered the surface of the MEW scaffolds uniformly, which verified the feasibility of GelMA HFM cross-linking in the ammonium sulfate solution. As shown in Figure 4(d), the porosity and pore size distribution of the MEW scaffold and composite scaffold before and after cross-linking were analyzed by Image-J software. Among them, the porosity of the MEW scaffold is about 65.99%, the composite scaffold before cross-linking is about 37.26%, and the composite scaffold after cross-linking is about 40.38%. Through the analysis of the pore size of the scaffold, the pore size of the scaffold is mainly concentrated below 100µm ² . However, it is found from the figure that after the composite scaffold is cross-linked, a part of the pore size of the scaffold increases, because after the freeze-drying process, the moisture in the fiber evaporates, which causes the fiber to become thinner, which increases the pore size and porosity of the scaffold, but the pore size still concentrated below 100µm ² . This composite scaffold with pore size is similar to the extracellular matrix in morphology and structure, which is conducive to cell adhesion. ²⁰

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Figure 4.

Morphology characterization and pore size of scaffolds. (a) MEW scaffold; (b) composite scaffold before cross-linking; (c) composite scaffold after cross-linking. (d): (I) pore size distribution of composite scaffold before cross-linking, (II) pore size distribution of composite scaffold after cross-linking.

Biocompatibility analysis of scaffolds

To evaluate the composite scaffolds' biocompatibility composed of GelMA HFM and MEW scaffold, we inoculated human umbilical vein endothelial cells (HUVES) on three groups of scaffolds, namely, MEW scaffolds, coated scaffolds, and composite scaffolds. The attachment, viability, proliferation, and morphology of cells were investigated separately during a 7-day culture period. As shown in Figure 5(a), the cell proliferation analysis was performed on day 1,4 and 7 by cell count kit (CCK)-8. The number of cells on the MEW scaffolds were the least during the 7-day culture period, the count of cells on the coated scaffolds was the largest the day4, but not apparent increase at day7. Although the number of cells in the composite scaffolds was lower than that in the coated scaffolds on the first four days, the composite scaffolds showed signs of being more extensive than the coated scaffolds on the seventh day and were much larger than the MEW scaffolds. It was further confirmed that the GelMA HFM could promote cell adhesion and proliferation. Figure 5(b) shows that the cell viability was analyzed using live/dead experiments to assess the three groups of scaffolds' biocompatibility. Most of the cells were alive (green), and a few were dead (red). The cells' growth unevenly on the MEW scaffold, and the scaffold was not overgrown, due to the low efficiency of cell seeding and uneven planting caused by the large pore size of MEW scaffolds, and it was difficult for cells across the pores of scaffolds to grow. The cells grew very well on the coated scaffold and soon covered the entire scaffold in Figure 5(c). Because coated scaffolds were enveloped by GelMA hydrogel, increase the contact area of cells, most cells were adhered to at planting. However, it was difficult for the cells to grow inside the scaffold due to the hydrogel's compactness, and they were only growing on the hydrogel of the scaffolds. With the increase in the number of cells, the limited space on the scaffold surface makes it difficult to continue the cell proliferation. In Figure 5(d), the GelMA HFM covers the MEW scaffolds' surface, which reduces the pore size of the scaffolds and simulates the ECM to some extent. The cells can grow into the scaffold along the GelMA HFM and forming the three-dimensional structure by cells migrate to the MEW scaffold gradually. The composite scaffolds can maintain not only the structure but also the similarity with the morphology of the extracellular matrix in pore size, showing excellent biocompatibility and becoming an ideal candidate for tissue engineering scaffolds.

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Figure 5.

Cell proliferation and viability analysis. (a) Comparison of cell proliferation between three groups of scaffolds; (b) cell viability of the MEW scaffolds on day 1 and day 7; (c) cell viability of the coated scaffolds on day 1 and day 7; (d) Cell viability of the composite scaffolds on day 1 and day 7.

As shown in Figure 6, we could observe the cell morphology on three groups of scaffolds by confocal fluorescence microscopy. In Figure 6(a), cells growing in the MEW scaffolds could form the three-dimensional structure. However, it was difficult for the cells to grow all over the scaffolds because of the large pore size of the MEW scaffolds, which seriously hinders applying MEW scaffolds in tissue engineering. Figure 6(b) shows the growth of cells on the coated scaffolds, although the scaffolds showed excellent biocompatibility, the surface of the scaffolds were enveloped by GelMA hydrogel, it was difficult for the cells to migrate to the internal MEW scaffolds. In Figure 6(c), the composite scaffolds almost covered the advantages of MEW scaffolds and coated scaffolds, which not only possessed excellent biocompatibility but also can form the three-dimensional structure. The GelMA HFM covers the MEW scaffolds' surface, the cells can migrate to the MEW scaffolds along the GelMA/PEO fibers, and the whole scaffold can be overgrown, quickly. The further demonstrated that the composite scaffolds could improve seeding efficiency and excellent biocompatibility. The above experimental results show that by combining MEW and electrospinning technology, GelMA HFM is covered on the surface of the MEW scaffold, which significantly reduces the pore size of the scaffold surface, increases the inoculation area of the scaffold and cells, and increases the cell size of the scaffold. Inoculation efficiency. Moreover, it can create a three-dimensional microenvironment. Cells migrate to the bottom MEW scaffold through GelMA fibers, which they would grow up in a 3D microenvironment rather than only on a plain surface.

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Figure 6.

Cell morphology analysis on day 7. (a) Cell morphology of PCL scaffold; (b) cell morphology of the coated scaffold; (c) cell morphology of the composite scaffold.