Page 40 - IJB-8-2

3D-Printed β-TCP scaffolds promote Osteogenic Differentiation of BMSCs in an m6A-Dependent Manner
1. Introduction transcriptional factor downstream of RUNX2 . Notably,
[13]
RUNX2 is also regulated by N6-methyladenosine (m6A)
Bone defect is one of the most common diseases RNA methylation. Yan et al. discovered the dual signaling
encountered in the field of orthopedics. With the cascades of osteogenic pathways: (i) Methyltransferase-
development of tissue engineering, allochthonous like 3 (METTL3) upregulates m6A methylation of
and autogenous bones have commonly been used RUNX2, which increases RUNX2 stability and level,
for transplantation into the site of bone defects for and contributes to osteogenesis; and (ii) METTL3
therapeutic purposes . However, this never addresses
[1]
the problems, such as a shortage of bone sources and increases m6A methylation of pre-miR-320, decreases
high infection rates. In view of the limitation of bone miR-320 levels, upregulates RUNX2 levels, and
improves osteogenesis . The dual mechanism of m6A
[14]
sources, degradable biomaterials such as hydroxyapatite modification on osteogenic differentiation may be caused
(HA) and β-tricalcium phosphate (β-TCP) have become by high or low levels of m6A modification . However,
[15]
alternative choices for use in bone repair. β-TCP has
been widely applied in clinical treatments due to their it is still unknown if the upregulation of RUNX2 after
promising results. β-TCP has several beneficial properties β-TCP stimulation is related to m6A modification.
in the aspects of biocompatibility, osteoconductivity, M6A RNA methylation plays an important role in
osteoinductivity, and biodegradability, and can be easily the regulation of numerous cell behaviors. M6A works
manufactured into porous structures, which further through three mechanisms: (i) Writing, which is regulated
improves its biodegradability and makes it suitable as by a methyltransferase complex consisting of METTL3,
a bone substitute for clinical application. Numerous METTL14, Wilms tumor-1-associated protein (WTAP),
clinical trials have shown that β-TCP has a similar effect and other methyltransferases, also known as writers [16-18] ;
on bone repair as allografts . Conventionally, β-TCP is (ii) erasing, which is regulated by demethylases fat mass
[1]
used in the shape of disks or granules. Three-dimensional and obesity-associated protein (FTO) and AlkB homolog
[19]
(3D) printing technology, as an emerging technology, 5 (ALKBH5), also known as erasers ; and (iii) reading,
has provided a new prospect in the treatment of bone which is regulated by m6A-binding proteins, including
defect. 3D printing can be used to make porous β-TCP, YTH family proteins and IGF2BP family members, also
which is beneficial for ingrowth of new bone. In addition, known as readers. M6A also influences the osteogenic
β-TCP combined with bone marrow mesenchymal stem differentiation of BMSCs in different ways; however, there
cells (bone marrow stem cells [BMSCs]) has shown are contradictory opinions regarding the m6A regulation
significantly higher degree of new bone formation mechanism. Wu et al. found that parathyroid hormone
compared to β-TCP alone . β-TCP/BMSCs combination (PTH)-induced osteogenic effects and the translation
[2]
could be used to treat bone defects, bone nonunion, and efficiency of parathyroid hormone receptor-1 (Pth1r)
[20]
other orthopedic diseases requiring bone implantation, mRNA were decreased after the knockout of METTL3 .
with a lower infection rate and a higher success rate of In contrast, METTL3 positively regulates myeloid
artificial bone implantation [3-7] . The osteoinductivity of differentiation primary response 88 (MYD88), activating the
β-TCP is probably related to the osteogenesis induced nuclear factor kappa B (NF-κB) signaling pathway, which
by BMSCs. It has been suggested that β-TCP promotes is regarded as an inhibitor of osteogenesis. Meanwhile, the
upregulation of osteocalcin (OCN), osteopontin (OPN), adverse effects of METTL3 can be reversed by ALKBH5 .
[21]
bone sialoprotein (BSP), and bone morphogenetic M6A modification also regulates interaction between cell
protein 2 . Moreover, Runt-related transcription factor and extracellular matrix. For example, overexpression
[8]
2 (RUNX2), the key transcription factor in osteogenesis of METTL3 promotes accumulation of ECM in human
of BMSCs, was also increased in expression level after Tenon’s capsule fibroblasts . However, the relationship
[22]
7 days of TCP stimulation . However, the mechanism between osteoinductivity of β-TCP and m6A modification
[9]
by which β-TCP contributes to the high expression of remains unclear.
RUNX2 in BMSCs is not fully understood. Therefore, the aim of this study is to prove that
RUNX2 is an indispensable transcriptional factor β-TCP might promote the osteogenesis of BMSCs by
for the commitment of mesenchymal stem cells toward upregulating RUNX2 in an m6A-dependent manner,
the osteoblast lineage . Knockout of RUNX2 causes further providing new evidence to support the clinical use
[10]
osteogenesis blockage at the cartilage stage, suggesting of β-TCP in bone defect treatment.
that RUNX2 is a promoter of early mineralization .
[11]
Mechanistically, RUNX2, as a transcriptional factor, can 2. Materials and methods
activate a large number of bone-related genes, including 2.1. 3D printing of β-TCP scaffolds
OCN, OPN, bone sialoprotein, and alkaline phosphatase
(ALP) . The osteoblast differentiation program also 3D printing (REGENOVO, China) was utilized to make
[12]
requires the activation of the genes like osterix to encode a β-TCP scaffolds for implanting the β-TCP into Sprague-
32 International Journal of Bioprinting (2022)–Volume 8, Issue 2