Page 189 - IJB-9-2

International Journal of Bioprinting A regulated GelMA-MSCs scaffold by three-dimensional bioprinting

negative control group, and microRNA-410-mimics group The nozzle specification was 25 G, and the moving speed
were stained with Ca-AM/PI live and dead reagent (sample was 360 mm/min. After printing, the GelMA hydrogel was
size = 9). For the specific staining and culture methods, see fully cured after irradiation with 405 nm, 3 W light source
the detailed procedures set out in the above. After staining, for 30 s, and GelMA hydrogel scaffold was finally obtained.
the fluorescent pathway was observed under the confocal
microscope, and the living and dead cells were counted 2.5.2. Surgical procedure and experimental design
with ImageJ software in the later stage. All animal experiments were approved by the Medical
Ethics Committee of Soochow University. All animal
2.4.5. Chondrogenic differentiation promotion assay
manipulations were performed in accordance with the
Alcian blue staining was used to detect whether Guide for the Care and Use of Laboratory Animals of
microRNA-410 promoted chondrogenic differentiation Soochow University. A total of 18 3-month-old New
of MSCs. MSCs of passage 3–5 were transferred into Zealand rabbits weighing approximately 3 kg were
chondrogenesis medium for further chondrogenic studied. Eighteen New Zealand rabbits were randomly
differentiation experiments. The medium was changed divided into three groups: (i) blank group (defect
every 2 days, and Alcian blue staining was performed sutured without any treatment); (ii) GelMA-MSCs group
on days 14 and 21, and the medium was removed. After (GelMA scaffolds containing MSCs transplanted into the
washing with PBS for 3 times, paraformaldehyde fixative defect); and (iii) GelMA-microRNA-410-MSCs group
solution was added for 15 min for full washing and drying. (GelMA scaffolds containing MSCs with upregulated
Alcian blue staining solution was added for 30 min and microRNA-410 transplanted into the defect). The rabbits
then removed for cleaning, followed by the addition of were anesthetized with sodium pentobarbital injected into
Alcian blue complex dye solution which was for 60 s. the ear vein. The skin inside and outside the knee joint
After washing with PBS, the solution was placed under was sterilized, and a longitudinal incision of about 2 cm
an inverted microscope. The degree of chondrogenic in length (i.e., one finger’s distance outside the patella) was
differentiation was observed. made in the knee patella. The subcutaneous tissue, muscles,

2.5. In vivo evaluation of cartilage defect repair by and tendons were isolated, and the patella subluxation was
3D-printed scaffolds pushed laterally after internal examination to expose the
articular surface of the distal femoral condyle. A 5.0 K-wire
2.5.1. 3D bioprinting of the GelMA-MSCs scaffold drill with an electric drill was installed to a depth of 5 mm.
About 80% adherent cells were selected, digested, and All animals returned to normal diet after operation, and
centrifuged. The supernatant was removed, and the pellet ceftiofur (5 mg/kg) was injected to fight infection on the
nd
was, then, suspended with GelMA solution. In traditional 2 day after operation. The wounds were observed every
printing, GelMA bioink is often put into the syringe 3 days and the dressing was changed. Three animals in
directly until it becomes gel-like. In the present study, the each group were sacrificed at weeks 6 and 12.
GelMA solution was added to a print syringe and stored 2.5.3. Computed tomography surface reconstruction
in a −20°C refrigerator for 6 – 8 min, away from light, to analysis
make the GelMA turn into a gel more quickly. This step can
make the printed GelMA bioink more formable and the At weeks 6 and 12, animals of the corresponding group
printing process smoother. According to physicochemical were sacrificed by air embolization introduced from the
properties for low-temperature curing of GelMA, the ear vein. After disinfection, the soft tissues were dissected,
nozzle of 3D bioextrusion printer (BP6601 - EFL) was set and the distal femur was dissected. Care should be taken to
at 25°C and placed in a syringe for 5 min. The temperature avoid damage to the integrity of the distal femoral condyle.
of the printer platform was adjusted to 5°C, with the The removed distal femoral condyle specimens were
air pressure adjusted to 10–30 kPa. After the hydrogel immersed in a centrifuge tube containing 10% formalin
microfilaments were stably extruded from the nozzle, the for 48 h. The fixed distal femoral condyle specimen was
hydrogel microfilaments were printed on the base plate. The taken out, and the computed tomography (CT) scanner
height of the printing scaffold was 5 mm and the diameter was turned on and warmed up for 30 min. The scanning
was 5 mm, and the average number of cells per scaffold parameters were set to 70 kV, 141 mA, and 1750 ms, and
5
was approximately 5 × 10 . It was a 5 mm cylinder with a the spatial resolution was 18 μm. The hatch door was
height of 0.2 mm for each layer. 1 × 10 cells were mixed opened to take out the foam box. Each pair of specimens
7
with 5 mL GelMA solution and pipetted evenly. Eight was placed side by side at the designated position in the
GelMA-MSCs scaffolds and eight GelMA-microRNA-410- foam box. The hatch door was closed for scanning, and the
MSCs scaffolds were made for subsequent experiments. operation was repeated until all specimens were scanned.

Volume 9 Issue 2 (2023) 181 https://doi.org/10.18063/ijb.v9i2.662