Page 194 - IJB-9-3

International Journal of Bioprinting 3D-printed vascularized biofunctional scaffold

by their limited sources and the need for surgical bone some positively charged growth factors such as VEGF to
extraction, which may lead to complications such as nerve form strong electrostatic bonds with its surface, which can
damage and infection [2,3] . Allogeneic bone grafts could slow down the release of growth factors or other drugs .
[25]
cause adverse conditions such as immune reactions, failure The addition of Lap to hydrogels has also been shown
to integrate with the host bone, and slow remodeling . to affect the mechanical properties of hydrogels . In
[25]
[4]
A 3D-bioprinted active scaffold can overcome these addition, Lap has been reported to modulate the immune
limitations and meet the needs of anatomical remodeling microenvironment and promote bone defect repair
and functional repair of bone defects . However, there are through the release of Mg and Si 4+[26,27] . These advantages
2+
[5]
still many challenges in the application of 3D bioprinting inspired us to conclude that combining GA hydrogel, Lap
technology for clinical bone defect repair, and the selection nanoparticles, and PRP may be a promising combination
of bioinks with strong pro-vascularization ability and strategy to achieve enhanced therapeutic effects of PRP by
osteoinductive bioactivity is the first challenge that needs slowing the release of various growth factors.
to be overcome . In the present study, we constructed a PRP-GA@
[6]
Osteogenic inducing factors represented by bone Lap composite bioink and demonstrated its function of
morphogenetic protein 2 (BMP-2) and pro-angiogenic sustained slow release of various growth factors. Moreover,
growth factors represented by vascular endothelial growth we investigated its effects on the proliferation, migration,
factor (VEGF) are expensive and physicochemically differentiation, and tubule formation of human umbilical
unstable, and could lead to complications such as ectopic vein endothelial cells (HUVECs) and rat bone marrow
ossification and tumorigenesis, limiting their application in mesenchymal stem cells (BMSCs) by in vitro experiments
bone tissue engineering [7-9] . Finding an alternative, effective and demonstrated that it promotes macrophage M2
and safe “activating factor” that can be incorporated into polarization. This composite bioink was then printed layer-
3D-printed active bone repair scaffolds is important. by-layer with polycaprolactone (PCL) to construct a bone
Platelet-rich plasma (PRP) is a platelet concentrate obtained repair scaffold using 3D printing technology. By implanting
by centrifugation of whole blood from animals or humans, this scaffold subcutaneously and at the site of femoral
which, when activated, releases multiple growth factors [10,11] . condylar defects in rats, we found that this bioactive
The proportions of the various growth factors released by scaffold promotes rapid vascular growth into the scaffold
PRP match the normal proportions present in the body, and accelerates bone regeneration. This work demonstrates
allowing for optimal synergy of each growth factor . that PRP-based 3D-printed vascularized bioactive scaffolds
[12]
have great potential for clinical translation in the treatment
During normal fracture healing, VEGF expression of bone defects.
typically peaks on days 5–10 after limb injury, while BMP-
2 expression continues to increase until day 21, suggesting 2. Materials and methods
the need for delivery systems that support the sustained
release of growth factors for long period of time [13-16] . 2.1. Primary culture of rat BMSCs
However, once PRP is activated, its multiple growth factors The bilateral femurs of 3-week-old male Sprague Dawley
are all released in a short burst, which is detrimental to the (SD) rats were quickly removed in a sterile environment,
repair of bone defects [17,18] . taking care to keep the marrow cavity closed. Then,
ophthalmic scissors were used to cut the bone marrow
Methacrylated gelatin (GelMA) and methacrylated cavity open at both ends on an ultraclean bench, and the
alginate (AlgMA) have good biocompatibility and can bone marrow cavity was rinsed four to six times with
form a uniform and stable pore-like structure within the α-MEM (Gibco) complete medium and placed in a 10-mL
gel after light-curing crosslinking, and a mixture of the two Petri dish using a sterile syringe. Then, the cells were placed
has better printability and mechanical properties [11,19-21] . in an incubator for 36–48 h of static culture. Nonadherent
In addition, bioactive molecules can be encapsulated in cells were carefully removed and the medium was replaced.
GelMA/AlgMA (GA) hydrogels and released slowly by When the cells grew to 85%–90% confluence, they were
diffusion, thus prolonging their retention time in bone passaged, and the third or fourth passaged cells were used
defect sites . On the other hand, nanoclays, such as for subsequent experiments.
[22]
laponite (Lap), have emerged as a new class of biocompatible
materials with strong drug loading capacity and have 2.2. Preparation of PRP and hydrogel precursor
potential to become strength-enhancing additives [23,24] . solution
Lap is a disk-shaped nanoparticle with a diameter of about PRP was prepared from the whole blood of rats after systemic
25 nm and a thickness of about 1 nm. The negatively heparinization by two centrifugations to remove serum
charged surface and positively charged edges of Lap allow and red blood cells. GelMA and AlgMA were dissolved in

Volume 9 Issue 3 (2023) 186 https://doi.org/10.18063/ijb.702