Page 246 - IJB-10-5

International Journal of Bioprinting 3D printed hydrogels for tumor therapy

37°C. After culturing for 1, 3, and 7 days, cell proliferation expressed as the mean ± standard deviation (SD). One-way
was evaluated using the MTT assay. analysis of variance (ANOVA) was performed to carry out
To investigate the effect of Mg release on rBMSCs statistical analysis. Data analyses in the current study were
2+
osteogenic differentiation, 1 × 10 cells per well were conducted using Excel and SPSS.
5
seeded on hydrogel samples in a 24-well cell culture plate. Differences were considered statistically significant
Following a culture period of 3 days, the culture medium when p < 0.05, p < 0.01, and p < 0.001.
was replaced by an osteogenic differentiation medium
(which was prepared by adding 0.1 mM dexamethasone, 50 3. Results and discussion
mM ascorbic acid, and 10 mM β-sodium glycerophosphate 3.1. Synthesis and characterization of MgHAp,
in culture medium). After culturing for 7 and 14 days, GelMA, and PDA@DOX
the activity of alkaline phosphatase (ALP) of rBMSCs Previous studies have employed biomimetic self-assembly
on the hydrogel samples was measured using an ALP mineralization method to fabricate HAp to mimic the
assay (Wako, Japan) according to the manufacturer’s structure and composition of natural bone. As a result,
39
protocol. Additionally, the total protein concentration was HAp synthesized through biomineralization process
measured using the BCA kit (Thermo Fisher Scientific). exhibited a positive effect on cell activities (e.g., cell
Moreover, the expression of ALP was assessed by means attachment, proliferation, and differentiation) and could
of ALP staining. Briefly, after being cultured in osteogenic promote new bone formation. Moreover, considering the
differentiation medium for 14 days, rBMSCs were fixed inherent components of natural bone, i.e., inorganic HAp,
with 4% paraformaldehyde (Sigma-Aldrich, St Louis, MO, organic COL1 and citric acid, research employing COL1
USA) for 15 min, washed with PBS, and incubated with and citric acid as bitemplate for HAp nanocomposites
ALP staining solution for 30 min in the dark. The images synthesis emerged as COL1 enabled to modulate
were recorded using a microscope (Leica DMi8, Germany).
nucleation sites and crystal growth of HAp due to its well-
Furthermore, the expression of osteogenesis-related organized fiber orientation, while citric acid could regulate
genes, such as osteocalcin (Ocn), Col1, runt-related HAp formation owing to its abundant carboxyl groups. 35,40
transcription factor-2 (Runx2), and bone morphogenetic Therefore, in the current study, HAp nanocomposites with
protein-2 (Bmp2) was studied using quantitative real-time a great similarity in structure and composition of apatite
polymerase chain reaction (qRT-PCR) analysis. Briefly, in natural bone were synthesized using COL1 and citric
after culturing rBMSCs on hydrogel samples in osteogenic acid as bitemplate through a biomineralization process.
medium for 14 days, the total RNA was extracted using Magnesium is an important metal element in human body,
Trizol reagent (Servicebio, China). The obtained RNA with 60% present in the human bone. Studies have indicated
was reverse-transcribed to complementary DNA (cDNA) that Mg played an essential role in bone metabolism
2+
using the Revert-Aid First Strand cDNA Synthesis Kit by regulating osteoblast and osteoclast activities. 36,41
(Thermo Fisher Scientific). Then, qRT-PCR analysis Moreover, compared to HAp, MgHAp could promote
was performed. Housekeeping gene glyceraldehyde cell proliferation and osteogenic differentiation through
3-phosphate dehydrogenase (Gapdh) was used as the activating integrin on the cell surface. Thus, MgHAp
42
control to normalize the expression of osteogenesis-related was synthesized in the current work for investigation. As
genes. The 2 −ΔΔCt method was used to calculate the mRNA shown in Figure 2A, the morphology of both HAp and
expression levels. The sequences of the primers ae listed in MgHAp nanocomposites exhibited a plate-like structure,
Table S1, Supporting Information. and the size of HAp and MgHAp nanocomposites was
In addition, after being cultured in osteogenic medium around 40–70 nm, showing great similarity to the shape of
for 14 days, rBMSCs on hydrogel samples were fixed with apatite in natural bone. Figure 2B and C show the element
4% paraformaldehyde for 15 min and then incubated with compositions of HAp and MgHAp nanocomposites.
anti-COLI rabbit primary antibody (Abcam) for 1 h and Notably, there was a small amount of Mg element in the
then FITC-conjugated goat anti-rabbit secondary antibody EDS spectrum, as shown in Figure 2C, indicating the
2+
(Santa Cruz Biotechnology Inc., USA) for 1 h. Cell nuclei presence of Mg in MgHAp nanocomposites. As shown
were stained with DAPI solution. The morphology of in Figure 2D, XRD patterns showed that HAp and MgHAp
rBMSCs was examined using a confocal laser scanning nanocomposites were mainly hydroxyapatite phase.
microscope (Leica DMi8, Germany). However, due to the incorporation of COL1 and citric acid
in the nanocomposites, HAp and MgHAp showed a low
2.9. Statistical analysis crystallinity in comparison to pure hydroxyapatite mineral.
The results presented in the current study were obtained Furthermore, compared to HAp nanocomposites, MgHAp
from at least three separate samples or tests and are nanocomposites exhibited an even lower crystallinity due

Volume 10 Issue 5 (2024) 238 doi: 10.36922/ijb.3526