Page 567 - IJB-9-6

International Journal of Bioprinting Osteogenic, antibacterial CpTi-MgOCu implants

3.2. Histological analysis and histomorphometry: presence, and osteoblast recruitment at the bone–implant
Bone-implant interface interface. Across all compositions, mature bone formation
To assess the biological performance of the compositions with well-embedded osteocytes and focal outward growth
in a physiological environment, an in vivo rat model was of osteoblastic regions from the implant surface can be
utilized. CpTi was considered the control, while CpTi- observed. CpTi’s trabecular bone formation was followed by
MgO and CpTi-MgO-Cu were treated as the treatment a thick osteoid lining at the BIC. In contrast, both CpTi-MgO
compositions. Figure 3 presents bone sections of porous and CpTi-MgO-Cu compositions exhibited newly formed
implants stained by Gomori’s trichrome, H&E, and SRBS trabecular bone directly apposed to the outer surface of the
with approximately 40 vol.% porosity. H&E-stained implant, indicating superior osseointegration performance
bone sections (Figure 3b) were observed for possible compared to CpTi. Furthermore, the SRBS-stained histology
inflammatory markers. None of the compositions show any micrographs reveal a higher degree of mature bone infiltration
inflammatory response, including neoplasia and necrosis . in the porous channel for CpTi-MgO and CpTi-MgO-
[51]
Gomori’s trichrome stain was employed to evaluate muscle Cu than CpTi. To obtain a clearer idea on the remodeling
fiber formation and collagen presence at the BIC (Figure 3a). process undergone at the BIC, SRBS-stained histology was
At the BIC, all three compositions exhibited interwoven employed for histomorphometric analysis to evaluate the
muscle fibers within collagenous regions, indicating early- mature bone formation at the BIC (Figure 3c) within the
stage osseointegration and mineralization front. Notably, specified ROI of 100 µm. Among the tested compositions,
these regions appeared visibly thinner for CpTi than CpTi- CpTi-MgO demonstrated the highest amount of matured
MgO and CpTi-MgO-Cu, suggesting a higher degree of bone formation at the BIC (49.5 ± 11.5%), followed by
osteogenesis in the latter compositions attributed to MgO. CpTi-MgO-Cu (38.2 ± 7.2%), while CpTi exhibited the
Further examination at higher magnification revealed gaps least amount (12.1 ± 9.2%). This suggests a higher affinity
at the BIC for CpTi, whereas for CpTi-MgO and CpTi- and enhanced biological response of the host tissue toward
MgO-Cu, these gaps were filled with osteogenic fronts the chemical makeup of CpTi-MgO and CpTi-MgO-Cu.
accompanied by small areas of muscle fiber presence. Although the mature bone formation between CpTi-MgO
Compared to CpTi-MgO and CpTi-MgO-Cu, the pink- and CpTi-MgO-Cu falls within the error range of each other,
bluish regions at the BIC for CpTi-MgO indicated the the difference in mean values can be attributed to the delayed
presence of muscle fibers interwoven with collagenous osseointegration observed in the latter composition due to
regions. Conversely, the bright pink regions at the BIC for the presence of Cu, as discussed earlier.
CpTi-MgO-Cu indicated the presence of muscle fibers
without collagen, suggesting delayed bone maturation and 3.3. Infection prevention
[52]
osseointegration due to the presence of Cu. CpTi does not possess inherent antibacterial capability .
In order to address post-surgical infections, the addition of
The observations described above are further Cu was implemented in the CpTi-MgO composition. Cu
supported by the H&E-stained histology micrographs is well-known for inhibiting bacterial growth through the
(Figure 3b), where varying shades of pink and purple on-contact killing of bacterial cells. Since S. aureus is one
reveal distinct demarcations representing mineralized of the most commonly occurring infections in vivo , the
[53]
bone, osteoid lining, and osteoblast recruitment regions. antibacterial efficacy of the CpTi-MgO-Cu material against
In the case of CpTi, only certain regions exhibited a this bacterial strain at 24, 48, and 72 h time points using
mineralized bone front at the BIC, while it was absent CpTi as the negative control was evaluated (Figure 4a). After
in other regions. In contrast, CpTi-MgO visibly 24 h of bacterial culture, we observed a significant reduction
demonstrated a higher presence of mineralized bone in bacterial viability on the agar plate for CpTi-MgO-Cu.
directly at the BIC, followed by an osteoid mineralization Bacterial colony counting on the agar plate showed a 95%
front, indicating superior early-stage osteogenic antibacterial efficiency for CpTi-MgO-Cu compared to CpTi.
performance. Upon closer examination at the BIC, SEM images taken after 24, 48, and 72 h of culture revealed
CpTi-MgO revealed the infiltration of mineralization a significant reduction in planktonic bacteria on the surface
fronts into the implant area, encompassing the implant of CpTi-MgO-Cu. At 24 h, the bacterial inhibition efficacy
regions. However, H&E histology for CpTi-MgO-Cu evaluated from the SEM images showed a 57% reduction in
showed a lower degree of mineralized bone formation planktonic bacteria on the surface of CpTi-MgO-Cu. At 48
at the BIC compared to CpTi-MgO, although similar and 72 h, enhanced antibacterial efficacy was observed, with
implant area infiltration features into the implant area a 53 and 81% reduction in planktonic bacteria on the surface
were observed.
of CpTi-MgO-Cu, respectively. The SEM images at 48 and
SRBS-stained histological micrographs, shown in 72 h showed bacterial cells adhering to each other, which
Figure 3c, depict trabecular bone formation, osteoid resulted in septum formation on the surface of CpTi-MgO-

Volume 9 Issue 6 (2023) 559 https://doi.org/10.36922/ijb.1167