Page 140 - IJB-9-2

International Journal of Bioprinting Hybrid biofabrication of neurosecretory structures

2.5. Scanning electron microscopy (SEM) and compressive tests were performed. First, the mechanical
transmission electron microscopy (TEM) strength of the prepared PLLA/gelatin was evaluated and
According to the previous method , hybrid biofabricated compared to that of the PLLA nanofibers. The tensile
[25]
3D models were immersed in 3% pre-cooled glutaraldehyde strength and elongation deformation were observed
solution at 4°C overnight. Then, 3D models were and recorded over time. Then, with the simple hydrogel
dehydrated by a series of concentration gradients (30%, scaffold as the control, the compression modulus of the
50%, 75%, 90%, 95%, and 100%) of ethanol for 30 min per hybrid biofabrication model was measured, and the stress-
sample and dried at critical points. SEM observations were strain curve of the hybrid biofabrication model under the
performed after spraying platinum on the surface of the 3D corresponding pressure was observed.
model. The intracellular ultrastructure of the 3D models 2.9. Contact angles
was observed by TEM. Cell precipitation was fixed with
2.5% of glutaraldehyde and 1% of osmium acid at 4°C for The water contact angles of PLLA and PLLA/gelatin were
2 h. The samples were dehydrated with a series of acetone measured by a German OCA-20 video optical contact angle
solutions (30%, 50%, 70%, 80%, 90%, and 100%) and then meter. The contact angle was measured after stabilizing the
routinely infiltrated, embedded, sectioned, and stained. deionized water droplets (approximately 0.2 mL in PLLA
Images were acquired from a TEM (TECNAI 10, Philips, and PLLA/gelatin). Each group was repeated five times
Netherlands). and averaged. The hydrophilic properties of PLLA/gelatin
and PLLA were compared.
2.6. Western blotting
2.10. Animal experiments and pathology
Referring to the previous methods , Western blotting
[30]
was conducted to quantify the protein expression of the Twelve BALB/c nude mice (aged 6 – 8 weeks, 18 – 25 g) were
hybrid model. After culturing in 2D model, PLLA/gelatin used in the experiments. A 0.5-mm incision was made into
membrane scaffold, and AGH hydrogel, the expression the back skin of the nude mice in which the scaffolds of the
of PC12 cell-specific proteins MAP2 and tubulin-β hybrid biofabrication group were implanted. Six mice were
th
th
was quantified. Briefly, total protein was obtained and dislocated and killed on the 7 and 14 days, respectively.
quantified according to the kit instructions (KGPBCA, The subcutaneous grafts were dissected, fixed with 4% of
Nanjing). Samples were separated by sodium dodecyl- paraformaldehyde, embedded in paraffin after dehydration,
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and sliced into 5-μm sections for pathological staining. All
and then transferred to polyvinylidene difluoride (PVDF) animal experiments were conducted under the supervision of
membranes (100 V constant pressure, 40 min). After sealing the Animal Research Ethics Committee of the First Affiliated
in 5% of skim milk solution, the membranes were incubated Hospital of Anhui Medical University (PJ2020-12-07).
overnight with primary antibodies to MAP2 (Abcam) 2.11. Statistical analysis
and tubulin-β (Proteintech) in a 4°C shaker and further
incubated with peroxidase-conjugated secondary antibody The data were statistically analyzed and plotted by
(Southern Biotech) at 37°C for 1 h. Finally, the membranes GraphPad Prism 9.0.1 and Origin pro 2108 software, and
were detected through the ECL chemiluminescence the data are expressed as mean ± standard deviation (mean
method (Merck Millipore, German ECL kit). ± SD). t-test was used to analyze the statistical significance,
and statistical differences are expressed by *P < 0.05,
2.7. Enzyme-linked immunosorbent assay (ELISA) **P < 0.01, and ***P < 0.001.
The culture supernatants from the 3D and 2D cell culture
models were collected every day for 9 days. The secretion 3. Results and discussion
of noradrenaline (NE) and met-enkephalin (MEK) in the The advent of 3D-bioprinted tissue-like structures
culture supernatant of each group was determined by the in vitro marks the emergence of a new field for organ
conventional double antibody sandwich ELISA method. reconstruction, which is considered a new development
The absorbance value was read at 450 nm and the levels of direction with infinite application potential [34,35] . However,
NE and MEK in the culture supernatant were calculated 3D bioprinting of organs is still constrained by some
according to the standard curve. The specific steps were challenges. One of the challenges is the low accuracy of
conducted according to the instructions of the ELISA kit. extrusion bioprinting, mainly due to the complex thermal
and dynamic characteristics of bioinks, which make it
2.8. Stress-strain curve difficult to perform accurate bioprinting . Moreover,
[36]
To show the advantages of hybrid biofabrication in the poor mechanical strength of the tissue-like structure
terms of mechanical properties, mechanical tensile and of simple hydrogel scaffolds makes it difficult to maintain

Volume 9 Issue 2 (2023) 132 https://doi.org/10.18063/ijb.v9i2.659