Page 272 - IJB-10-3

International Journal of Bioprinting 3D bioscaffolds with SR1 for vasculogenesis

generated by following procedures; 75 mL of ethanol was core-shell bioinks were printed at 60 kPa (core) and 40
dissolved with 75 mg of blank nanoparticles. Additionally, kPa (shell) on a 37°C printing base with a crosshead speed
1 mL of dimethyl sulfoxide (DMSO) was dissolved with of 800 mm/min. The resulting sample dimensions were
25 mg of SR1. The above-prepared mixture of ethanol and approximately 40 × 40 × 0.6 mm, and it was incubated
blank nanoparticles was combined with the DMSO and at 37°C for an hour to allow the atelocollagen to gel.
SR1 solution. The resulting mixture was incubated for 3 h Subsequently, the sample was trimmed to a 5 mm diameter
under a vacuum state at a pressure of 100 bar. Then, the and immersed in PBS at 4°C to remove the Pluronic F-127.
pressure was lowered to 60 bar for a 30 min incubation.
Finally, 100 mg of SR1-laden nanoparticles were generated 2.5. In vivo studies
after drying for 12 h at a vacuum state. Characteristics of 2.5.1. Experimental animals
SMB 3, according to the manufacturer’s description, are Twenty-eight female Sprague-Dawley rats (age of 3
TM
as follows: a SiO content of 99%, an average pore diameter months, body weight of 240 ± 20 g) were purchased from
2
of 3.45 nm, an average BET surface area of >750 m /g, Koatech (Pyeongtaek, Republic of Korea) and housed in
2
a pore volume of >1.0 cm /g, and a tap density of ~0.12 a Sun Protection Factor environment of a 12 h light/12 h
3
g/cm . Scanning electron microscopy (SEM; SU8010, dark cycle. The animals had free access to water and food.
3
Hitachi, Chiyoda, Tokyo, Japan) was used to visualize the Animal experiments were approved by the Institutional
morphology and analyze the diameter of the nanoparticles.
Animal Care and Use Committee of Daegu-Gyeongbuk
2.3. Liquid chromatography-mass spectrometry (LC- Medical Innovation Foundation (Approval number:
MS) release test of nanoparticles KMEDI-22080801-01).
A QTRAP 6500+ low-mass LC-MS system (SCIEX, 2.5.2. Surgery and treatment
Toronto, Canada) was used to analyze the cumulative After a week of adaptation, the 28 rats were divided
release of SR1-laden nanoparticles. Calibration into three groups according to body weight: CT group
standard solutions were prepared by soaking SR1-laden (no treatment, n = 8), scaffold with blank nanoparticles
nanoparticles in deionized water (10 mg/mL) and diluting (NP@Sc) group (implanted scaffolds incorporated with
them with methanol to final concentrations of 10, 50, and blank nanoparticles, n = 8), and scaffolds with SR1-laden
100 part per billion. Samples were prepared by soaking nanoparticles (SNP@Sc) group (implanted scaffolds
SR1-laden nanoparticles in deionized water (10 mg/mL)
and releasing them after 2, 4, and 6 days. SR1-released incorporated with SR1 nanoparticles, n = 12). The animals
samples were filtered with a pore filter (pore size: 0.22 µm) were anesthetized by intraperitoneal injection of Zoletil
to remove the nanoparticles. The filtered solutions were (tiletamine/zolazepam) (30 mg/kg) and xylazine (10 mg/
subjected to centrifugation (10,000 × g, 10 min) to ensure kg). The rats then underwent surgery to create defects in
complete elimination of the remaining nanoparticles; their cranial bone (diameter = 5 mm) using a trephine bur
after centrifugation, the supernatants were diluted using and implant the scaffolds. Postoperatively, the rats were
methanol to a final concentration of 0.01 mg/mL. given subcutaneous injection of meloxicam (0.2 mg/kg) as
an analgesic. The animals were sacrificed for evaluation at
30
2.4. Scaffold fabrication 2 and 4 weeks after surgery.
Core-shell scaffolds were constructed using a coaxial
nozzle (inner needle 19 G, outer needle 14 G; Ramé- 2.5.3. Micro-computerized tomography analysis
hart, Succasunna, New Jersey, USA) equipped with a 3D Half of the animals in each group were anesthetized and
bioprinter (Root 1, Baobab Healthcare, Ansan, Republic of subjected to micro-computerized tomography (MCT)
Korea). For the shell, bioinks were prepared by combining scanning to evaluate bone regeneration and to visualize
3 w/v% neutralized atelocollagen (4°C; Baobab Healthcare, the whole cranial bone. The Quantum FX (PerkinElmer,
Ansan, Republic of Korea) and 3 w/v% alginate (viscosity Waltham, Massachusetts, USA) was used to perform MCT
>2000 cP, 20°C; Sigma-Aldrich, Burlington, Massachusetts, scanning 2 and 4 weeks after implantation.
USA) at a ratio of 3:1. Then, either SR1-laden nanoparticles 2.5.4. Microfil perfusion
or nanoparticles alone were mixed with the bioinks at a Four weeks after implantation, half of the animals in each
final concentration of 1 µM (SR1) or an equivalent weight group were perfused with Microfil compound (MICROFIL®
(nanoparticles only), respectively. MV-122; Flow Tech, Cheonan, Republic of Korea) to

Bioinks were prepared for the core by combining 40 evaluate blood vessel formation using MCT scanning. The
w/v% Pluronic F-127 (P2443, Sigma-Aldrich, Burlington, animals were anesthetized, and 0.2 mL of heparin (5000
Massachusetts, USA) with 100 mM calcium chloride, IU/mL) was injected intravenously into their tail vein. The
which served as a crosslinking agent for the alginate. The animals were then fixed on a polystyrene plate, and the

Volume 10 Issue 3 (2024) 264 doi: 10.36922/ijb.1931