Page 384 - IJB-9-5

International Journal of Bioprinting Vascularized bone regeneration

Transmission electron microscopy showed a nanoscale rate of SiO and Ca during degradation using ICP. The
2+
4-
4
mesoporous structure with an average mesopore size results showed that SiO was released slowly throughout
4-
4
of 3.833 nm (Figures S2 and S3 in Supplementary File). the entire testing period, while Ca was released suddenly
2+
These results indicate that we successfully fabricated in the first 2 days, followed by a slow release (Figure 3B
porous scaffolds with multiscale micro-nano structures and C). This constructed a vascularized bone regeneration
using 3D printing. microenvironment . We also measured the pH value
[23]
We also evaluated the mechanical strength of the PMBG, changes of the scaffold during degradation. The pH
PMBG/5TCP, PMBG/10TCP, and PMBG/20TCP scaffolds values of all scaffold groups remained between 7.1 and 7.5
(Figure 2D). The PMBG scaffold exhibited a compressive (Figure 3D), indicating that the microenvironment formed
modulus of 10.41 MPa and a compressive strength of by the scaffold degradation is suitable for cell growth and
1.07 MPa. In comparison, scaffolds containing rigid development. Meanwhile, after soaking the various groups
nanoparticles (TCP) exhibit higher compressive modulus of scaffolds in Tris-HCl, their surface mineralization
and ultimate compressive strength [34,35] . The maximum abilities were observed using SEM and X-ray EDS after
compressive strengths of PMBG/5TCP, PMBG/10TCP, 35 days, as shown in Figure 3E. Mineral particles were
and PMBG/20TCP scaffolds are 2.36, 9.4, and 4.68 MPa, detected on the surfaces of all scaffold groups. Notably,
respectively, and their corresponding compressive moduli the TCP-doped scaffolds demonstrated an elevated
are 18.61, 23.12, and 14.97 MPa, respectively (Figure 2E formation of mineral particles on their surfaces, along with
and F). The PMBG/10TCP scaffold exhibited the highest a corresponding increase in the accumulation of calcium
mechanical strength. We also tested the shrinkage rates and phosphorus. Importantly, this effect was found to
and printing accuracies of the scaffolds after sintering. be directly proportional to the degree of TCP doping.
The shrinkage rates in the transverse direction of the Therefore, considering all the advantages, PMBG/10TCP
PMBG, PMBG/5TCP, PMBG/10TCP, and PMBG/20TCP scaffolds were used for subsequent in vitro and in vivo
stents prepared by us are 39.89%, 37.86%, 34.90%, and experiments.
32.04%, respectively (Figure S4A in Supplementary 3.3. Biocompatibility testing of the scaffold
File). The shrinkage rates in the sagittal plane are 40.94%, To assess scaffold biocompatibility, a CCK-8 cell viability
38.07%, 34.81%, and 35.75%, respectively (Figure S4B assay was conducted to evaluate cell proliferation on
in Supplementary File). The printing accuracies of the each scaffold. As shown in Figure 4A, the proliferation
PMBG stent, PMBG/5TCP stent, PMBG/10TCP stent, and behavior of MSCs co-cultured with the scaffolds in each
PMBG/20TCP stent are 125.3, 158.67, 193.3, and 249.3 μm, group increased from day 1 to day 7. However, compared
respectively (Figure S5 in Supplementary File). Finally, to the control group, the proliferation rate of cells on the
using X-ray microscopy, we imaged the PMBG/10TCP PMBG and PMBG/TCP scaffolds slightly decreased. This
scaffold and found that it had interconnected pores and may be due to the effect of excessive calcium ion release
a regular morphology (Figure S6 and Videoclip S2 in in the first few days, which could have an impact on
Supplementary File).
cell proliferation . To further validate the reliability of
[36]
Additionally, we characterized the in vitro the CCK-8 results, a live/dead assay was conducted, as
degradation of PMBG, PMBG/5TCP, PMBG/10TCP, and shown in Figure 4B, where green indicates live cells and
PMBG/20TCP scaffolds. After 35 days, the degradation red indicates dead cells. In the co-culture of cells with the
rates of PMBG, PMBG/5TCP, PMBG/10TCP, and PMBG and PMBG/TCP scaffolds for 48 h, there were very
PMBG/20TCP were 39.17%, 29.53%, 24.19%, and 15.32%, few red fluorescent cells observed in all groups, indicating
respectively (Figure 3A). PMBG scaffolds degraded the that each scaffold possessed good biocompatibility.
fastest compared to the other scaffold groups, while the
degradation rate of the scaffold decreased with increasing 3.4. Osteogenic differentiation of cells on scaffolds
TCP doping ratio. This phenomenon may be attributed An experiment was conducted using ARS staining and
to the formation of more stable compounds between the ALP staining to assess the promotion effects of different
phosphate ions present in the TCP particles and the calcium scaffolds on in vitro osteogenesis of BMSCs. ALP staining
ions in the MBG, following the introduction of TCP into was performed on the 7th day, as ALP is a marker enzyme
the MBG system. The resulting increase in the stability of for early mature osteoblasts (Figure 4C). According to
the TCP-MBG composite could contribute to a reduction the study’s findings, the area of ALP staining was greater
in the degradation rate of the MBG material [30,33,35] , and this in the group treated with the PMBG scaffold compared
indicates that TCP doping can regulate the degradation rate to the control group. Additionally, the group treated with
of the scaffold, making it compatible with the rate of bone the PMBG/TCP scaffold showed the largest area of ALP
tissue regeneration. Furthermore, we measured the release staining compared to the other two groups. This was

Volume 9 Issue 5 (2023) 376 https://doi.org/10.18063/ijb.767