Page 121 - IJB-9-5

International Journal of Bioprinting DLP-printed scaffold for bone regeneration

Endochondral osteogenesis is the formation of limb achieve liquid-to-solid conversion under ultraviolet (UV)
bones, trunk bones, skull bones, etc. [12,13] . At the site of irradiation [38-40] . Numerous scholars have demonstrated
long bones, mesenchymal stem cells differentiate into that GelMA exhibited certain 3D printing properties
chondrocytes, which secrete cartilage matrix and form to promote the regeneration of bone tissue [41-43] . On the
cartilage template, followed by vascular invasion, cartilage basis of the above materials, in this study, we developed a
degeneration, and formation of ossification to complete kind of polymethacrylic acid (PMAA)-modified GelMA/
bone tissue regeneration [10,13,14] . Since the formation of PMAA bio-ink by digital light processing (DLP) printing
cartilage template and the invasion of blood vessels are two technique. The carboxyl functional group of PMAA
key processes of ECO, if blood vessels fail to regenerate in can facilitate the expression of HIF-1α by effectively
time after the ECO process is initiated by the chondrogenic chelating iron ions; thus, it could significantly promote the
differentiation of bone marrow mesenchymal stem cells chondrogenic differentiation of BMSCs via HIF-1α-SOX9
(BMSCs), ECO is likely to stall in the cartilage template and the regeneration of blood vessels via HIF-1α-VEGF.
stage, resulting in the failure of osteogenesis . Furthermore, the microporous structure of 3D-printed
[15]
scaffold actively promoted the growth of blood vessels and
Several pathways have been available to achieve the regeneration of bone tissue. This has created a promising
the differentiation of BMSCs to chondrocytes, with approach for the treatment of bone defects (Figure 1).
HIF-1α-SOX9 being an important one [16-18] . SOX9, a
significant regulator of cartilage differentiation, can 2. Materials and methods
effectively promote the differentiation and proliferation
of chondrocytes [19-22] . Apart from that, hypoxia-inducible 2.1. Materials
factor-1 alpha (HIF-1α) can also encourage the formation Gelatin from porcine skin was purchased from Sigma-
of vascular endothelial growth factor (VEGF) to effectively Aldrich, USA. Methacrylic anhydride (MA, 97%) and
stimulate vascular invasion, timely degeneration of lithium phenyl-2,4,6-trimethylbenzoyl phosphinate
cartilage, and regeneration of bone tissue [23-26] . Therefore, (LAP) were purchased from J&K, China. Methacrylic acid
HIF-1α is essential for the process of ECO. (MAA) was purchased from Aladdin Industrial, China.
Cell counting kit-8 (CCK-8), live/dead viability assay kit,
Iron ions and oxygen are known to be two necessary and phalloidin were purchased from Beyotime, China.
requirements for the degradation of HIF-1α by prolyl
hydroxylase domain (PHD). As a consequence, a decrease 2.2. Preparation of bio-inks
in the oxygen or iron ion concentration will contribute to Briefly, gelatin was dissolved in phosphate-buffered saline
a reduced ability of PHD, leading to an increase in HIF- (PBS) at 50°C under continuous stirring, and MA was
1α [27,28] . In recent years, many researchers have attempted added. After reaction in dark for 3 h, the products were
to induce the ECO process by adding desferrioxamine diluted with 2× PBS and then dialyzed against distilled
(DFO) to the material [29-31] . As a common iron chelator, water for 5 days at 40°C. The products were then filtered
DFO can promote the HIF-1α pathway, leading to with 0.22-μm paper filter and lyophilized, leading to a white
ECO. However, this exogenous factor will pose a major porous foam, before being stored at -20°C for further use.
biosafety risk if the problem of explosive release cannot
be solved [32-36] . Therefore, it becomes critical to explore GelMA and MAA were dissolved in PBS added with
materials to induce the ECO process from their own LAP (0.25% (w/v)) and tartrazine (0.05% (w/v)), then the
properties. Sun et al. prepared injectable hydrogels that can bio-inks composed of 15% GelMA/3% PMAA and 15%
imitate a hypoxic environment to achieve the regenerated GelMA/6% PMAA were prepared.
bone defects in rat femoral condyles through ECO using a
cell-free and growth factor-free strategy, which is a well- 2.3. Fabrication of scaffolds
established method to solve bone defects . However, The computer-aided design (CAD) model was designed as
[37]
although injectable hydrogels can match defect areas, a cylinder with interconnected pores, with a diameter of
they are difficult to be prepared directly as pre-designed 6 mm, height of 8 mm, and pore size of 600 μm. Then, a
macroscopic structures. It is therefore essential to use DLP printer (BP8601 Pro, EFL, Suzhou, China) was used
materials with three-dimensional (3D) printing property to prepare the scaffolds, and the parameters were adjusted
to fabricate bone tissue engineering scaffolds with desired for printing. Then, the scaffolds were strengthened under
structures. UV light for 3 min (Kernel parameters of GelMA scaffold:
layer height, 100 μm; light intensity, 10 mW/cm ; exposure
2
Methacrylated gelatin (GelMA) not only has similar time, 15 s; temperature, 29°C. Kernel parameters of
biocompatibility to extracellular matrix, but also GelMA/3% PMAA scaffold: layer height, 100 μm; light
2
possesses good photo-crosslinking ability which can intensity, 10 mW/cm ; exposure time, 13 s; temperature,

Volume 9 Issue 5 (2023) 113 https://doi.org/10.18063/ijb.754