Page 564 - IJB-9-6

International Journal of Bioprinting Osteogenic, antibacterial CpTi-MgOCu implants

demonstrate excellent in vivo biological performance and Serial dehydration was carried out in ethanol followed by
is used as a control. Adding MgO to CpTi is expected to embedment in polymethylmethacrylate (PMMA) . These
[39]
yield better early-stage osseointegration performance than embedded bone explants were cut on Ekakt saw into
TM
CpTi. PBF-fabricated porous implants with ~40 vol.% 200 µm thick sections, mounted on glass slides, and then
porosity were used for the study. ground to 20–50 µm thick sections using sand paper with
1200 grit size on Ekakt 400 micro grinder. The sections
2.4.1. Surgery and implantation procedure were then polished on the micro grinder using paper with
Male Sprague-Dawley rats with average weights between 4000 grit size. Gomori trichrome, hematoxylin & eosin
300 and 350 g were used for the in vivo study. The animals (H&E), and Sanderson’s Rapid Bone Staining (SRBS) were
were acclimatized in temperature- and humidity-controlled used to stain separate bone sections for each composition.
rooms in separate cages for at least 2 weeks. Buprenorphine The stained bone sections were imaged on a Keyence
(0.3 mg/kg) was subcutaneously administered to the animals digital microscope (Model VHX-7000, Itasca, IL, USA).
for alleviating pain 30 min before anesthesia. A prescribed H&E-stained slides were imaged for any visible markers
dose of IsoFlo® (isoflurane, USP, Abbott Laboratories, North that indicate an inflammatory response in areas around the
Chicago, IL, USA) coupled with oxygen (Oxygen USP, A-L implant. Gomori’s trichrome-stained slides were observed
Compressed Gases Inc., Spokane, WA, USA) was used to for the presence of muscle fibers and collagen at the bone–
anesthetize the animals. Once the animal’s movements ceased implant contact (BIC), and SRBS-stained slides for the
under anesthesia, the implantation area around the femur mineralized bone formation, osteoid presence at the BIC,
and the knee was shaved thoroughly and cleaned thrice and mineralization fronts.
with chlorhexidine and isopropyl alcohol scrubs. Lidocaine
hydrochloride (0.3 ml; without epinephrine) with a 0.5% Histomorphometric analysis was carried out using
concentration was subcutaneously administered as a localized SRBS-stained slides for each composition. To restrict the
numbing agent near the implantation area on each limb. The region of interest (ROI) to 100–150 µm from the implant
animal was then transferred onto a sterile surgery table area. surface, images were captured at 1000× magnification
A 2-inch incision was made along the femur on the lateral around the BIC region. Quantitative evaluation of
side above the distal femoral condyle. A unicortical defect of mineralized bone formation at the BIC was carried out
2.4 mm diameter was made on the lateral epicondyle using using Trainable Weka Segmentation in ImageJ with
gradually increasing drill bits and rinsed with saline to prevent Random Forest Algorithm for individual images [40,41] . At
thermal necrosis and remove bone fragments. The implant least seven regions were analyzed to quantify mineralized
was inserted, and the fascia and the skin over the incision bone formation at the BIC, which was expressed in %
were sutured with undyed braided coated with MONOCRYL- area fraction.
polyglactin 910 (Ethicon Inc., Somerville, NJ, USA). The
incision area was cleaned with saline scrubs and stapled. A 3. Results
similar procedure was carried out on the other limb of the CpTi shows good biocompatibility and no cytotoxicity.
animal. The animal was periodically monitored for its breathing Compared to bulk Ti6Al4V implants, CpTi is a more
rate during the surgery procedure. Lactated ringers solution popular coating material choice owing to the lack of
(LRS, 3 ml) for rehydration was subcutaneously administered strength and fatigue resistance. However, it is bio-inert
to the animal post-surgery, followed by meloxicam (0.2 mg/ and possesses no antibacterial capabilities. Besides, the
kg) administration as an anti-inflammatory analgesic, and surface properties of the implant influence its biological
monitored until the animal regained consciousness. Post- performance in the physiological environment. This
operative care was carried out for 3 days, with buprenorphine study aims to enhance the early-stage osseointegration
administration every 12 h and meloxicam every 24 h. The of CpTi by adding MgO and to induce inherent
animals were euthanized 6 weeks after the surgery by means antibacterial capabilities by adding Cu. This CpTi-
of carbon dioxide overdose, followed by cervical dislocation MgO-Cu material chemistry is expected to show
as a secondary measure, and the femoral bone with the metal superior biological performance in vivo compared to
implant was harvested. The Institutional Animal Care and Use CpTi and can be a potential metallic coating material of
Committee (IACUC) of Washington State University (WSU- choice for bulk metallic implants.
Pullman, WA)-approved protocol was followed to perform
the experimental and surgical procedure. 3.1. Microstructure and hardness
SEM micrographs of the etched surface for CpTi and CpTi-
2.4.2. Histological and histomorphometric analysis MgO (Figure 2b) show typical α’ martensitic needle-like
The bone–metal explants were fixed in 10% neutral structures typically observed in additively manufactured
buffered formalin for at least 72 h for tissue infiltration. CpTi due to the fast-cooling nature of the process .
[42]

Volume 9 Issue 6 (2023) 556 https://doi.org/10.36922/ijb.1167