Page 95 - IJB-9-3

International Journal of Bioprinting Biocompatible 3D printing photosensitive resin

Table 2. Resin hardness and impact strength performance of inhibitory effect on their proliferation at different time
points of the culture (Figure 6C and D). In addition, the
Sample Hardness (Shore D) Notched impact strength
(kJ/m ) different formulations of NIPUA showed less cell toxicity
2
PEGDA-0 84 (±2) 3.56 (±0.33) to L929 cells (Figure S4A–C). These data demonstrated
that NIPUA showed excellent biocompatibility.
PEGDA-4 80 (±1) 2.97 (±0.35)
PEGDA-8 81 (±1) 3.13 (±0.49) 3.10. Evaluation of the inflammatory effect of NIPUA
PEGDA-12 81 (±1) 3.01 (±0.39) in vivo and in vitro
PEGDA-16 81 (±1) 3.27 (±0.55) The biosafety of NIPUA was further evaluated in vivo.
The commercial resin and NIPUA were implanted
PEGDA-20 83 (±1) 3.74 (±0.09) into the back muscles of mice. After 7 days, the resins
PEGDA-24 79 (±2) 2.62 (±0.55) attached with muscle tissues were collected and subjected
to H&E staining. As shown in Figure 6A, the Trans and
spots on the resin’s surface. NIPUA showed the best alkali White groups caused severe muscular toxicity reactions.
corrosion resistance. The black spots that appeared on It demonstrated severe transverse muscle atrophy with
the surface of PEGDA-12 were smaller in size and less in varying degrees of hemorrhagic spots on the muscle edges,
number, which indicates that the NIPUA has better alkali varying thickness of myocytes, thin muscle bundles, and
corrosion resistance when the PEGDA content is 12 wt.%. partial cell necrosis [25,26] . The nuclei were densely packed,
with a large number of brown lipofuscin granules at
3.7. Dimensional accuracy test the ends of the nuclei (black arrows). No significant
The different sizes of microporous structures ranging from toxicological reactions were observed in the NIPUA group.
500 μm to 2 mm were designed and printed to test the Inflammatory reactions were present in all three groups,
dimensional accuracy of 3D printing. The test results are which were caused by the rejection of the organism to the
shown in Figure S3. The experimental results show that allogeneic graft contact. However, it is also obvious that
a microporous structure below 500 μm can be formed. In the inflammatory cell infiltration was more severe in the
addition, the smaller is the size of microporous structures Trans and White groups than in the NIPUA group. This
in different shapes, the greater is the impact on the indicated that NIPUA has good in vivo biocompatibility
size error and shape integrity. This is related to material and is not prone to toxic side effects.
refractive index, equipment resolution, and other factors.
It was demonstrated that macrophage M1 and M2
3.8. Hemocompatibility of NIPUA polarization are important during the inflammatory
The hemolysis test is used to evaluate the hemocompatibility responses. We further detected the inflammatory effect of
of biomaterials. According to ISO 10993-4, biomaterials NIPUA resins and commercial resins on Raw264.7 cells. LPS
with a hemolysis rate <5% are considered safe to use was administered as positive control. Pro-inflammatory
as blood-contacting materials. In the positive control factors including M1 markers IL-6, IL-1β, and TNF-α
group, blood cell membrane was disrupted, resulting were significantly increased in the Trans group and White
in hemolysis. The addition of PBS as negative control group compared to the NIPUA group (Figure 7B). While
did not result in hemolysis. The hemolysis rate of the the anti-inflammatory factors including M2 markers IL-10
resins with different PEGDA contents was less than 0.5% and TGF-β1 were significantly reduced in the commercial
(Figure 5A, 5B), indicating that the NIPUA has excellent resins group, NIPUA did not significantly affect the gene
hemocompatibility. expressions of inflammatory factors.

3.9. Biocompatibility of NIPUA to bone and 3.11. Preliminary investigation of the biosafety
muscle cells mechanism of NIPUA
According to the physiochemical evaluation, PEGDA-12 RNA sequencing was used to further explore the regulatory
in NIPUA was used for subsequent experiments. The mechanism of commercial resins in the cell cycle and
orthopedic surgical guide has unavoidable contact with apoptosis of MCET3-E1 cells. According to differentially
bone and muscle cells during surgery. The cytocompatibility expressed genes (DEGs) in Figure 8A, 1400 genes were
of NIPUA resin toward MC3T3-E1 bone cells and C2C12 upregulated while 1157 genes were downregulated
muscle cells was compared with commercial resin by cell in the NIPU group compared with the Trans group,
proliferation assay and live-dead assay. Trans and White whereas 901 genes were upregulated and 1234 genes were
commercial resin were extremely cytotoxic to MC3T3-E1 downregulated in the NIPU group compared to the White
and C2C12 cells. However, the NIPUA was cytocompatible group. Furthermore, several DEGs were enriched in the
with MC3T3-E1 and C2C12 cells as indicated by the absence cell according to the KEGG analysis (Figure 8B).

Volume 9 Issue 3 (2023) 87 https://doi.org/10.18063/ijb.684