Page 209 - IJB-10-5

International Journal of Bioprinting Biomimetic osteochondral scaffold

2.5. Isolation and cultivation of rat bone marrow and cartilage layer with FGF-18 in different amounts (1, 2,
mesenchymal stem cells 3, and 4 μg BMP-2 per g scaffold) was studied over a 30-day
Rat bone marrow mesenchymal stem cells (rBMSCs) were period. The BMP-2 ELISA kit (Westang, China) and FGF-
derived from the femoral cavity of 10 2-week-old Sprague– 18 ELISA kit (Westang, China) were used to determine
Dawley rats following the previous study. rBMSCs were the concentration of BMP-2 and FGF-18, respectively. The
36
cultured in DMEM with 10% FBS and antibiotics (100 U/ GF-loading amount in each group was defined as C . The
1
mL streptomycin and 100 U/mL penicillin) in an incubator cumulative release concentration at each time point can be
at 37°C with 5% CO . The medium was changed every 2 obtained and recorded as C day0 , C day3 , C day6 , ..., C day27 , C day30 .
2
days, and cells at three to five passages (P3–P5) were used Furthermore, the percentage release was calculated based
in all experiments. on the percentage of GF content to GF loading at a certain
time point. For example, the percentage of BMP-2 released
2.6. Alkaline phosphatase staining and collagen II on day 3:
immunofluorescence analysis
Rat bone marrow mesenchymal stem cells (rBMSCs)
seeded on tissue culture plates and cultured with osteogenic % release (day3 ) = C day3 ×100 % (I)
medium (DMEM with 10% FBS, 100 U/mL streptomycin, BMP−2 C
100 U/mL penicillin, 100 nM dexamethasone, 10 mM 1
β-glycerophosphate disodium salt hydrate, and 50 μM The released dose for every 3 days is calculated by
2-phospho-l-ascorbic acid trisodium salt) for 14 days were subtracting the release concentration at two adjacent time
set as the osteogenesis positive control group (OM-group). points. For example, the released dose of FGF-18 from day
After induction using different concentrations (0, 50, 100, 3 to day 6:
150, and 200 ng/mL) of BMP-2, samples were washed twice
with DPBS and then fixed with 4% paraformaldehyde (PFA)
for 20 min. The BCIP/NBT ALP kit (Sigma, USA) was Release dose FGF–18 (day 3 to 6) = C day6 – C day3 (II)
used for ALP staining and staining results were captured
using a macro camera (Canon, Japan). Furthermore,
rBMSCs seeded on tissue culture plates and cultured with 2.8. Release behavior of osteochondral scaffolds in
chondrogenic medium (DMEM with 5% FBS, 100 U/mL gelatin methacrylate gels
streptomycin, 100 U/mL penicillin, 10 nM dexamethasone, The integrated osteochondral scaffolds (3 μg/g BMP-2 of
50 μg/mL 2-phospho-l-ascorbic acid trisodium salt, 10 ng/ the subchondral layer and 2 μg/g FGF-18 of the cartilage
mL TGF-β1, and 1% insulin-transferrin-selenium) for 21 layer) were fabricated via cryogenic 3D bioprinting
days were set as the chondrogenesis positive control group (described in Section 2.3). The osteochondral scaffolds
(CM-group). After induction using different concentrations were then encapsulated into 5% GelMA gel to simulate the
(0, 10, 50, 100, and 150 ng/mL) of FGF-18, samples were in vivo matrix environment. The released content of BMP-
rinsed with DPBS twice, fixed with 4% PFA for 20 min, and 2 and FGF-18 were measured using ELISA kits (described
permeabilized with 0.2% Triton X-100 for 20 min. After in Section 2.7).
incubating in 1% BSA for 1 h, samples were treated with 2.9. Preparation and characterization of rat bone
collagen II (COL II) monoclonal antibody (Boster Biological marrow mesenchymal stem cell microspheres
Technology, China) diluted at 1:500 and incubated at 4°C To prepare the rBMSC microspheres, rBMSCs suspension
overnight. After rinsing with DPBS, samples were incubated was dropped into a microwell culture plate (Stemcell
in a solution of Alexa Fluor 488-conjugated secondary 5
antibodies diluted at 1:800 for 1 h at 37°C. The samples Technologies, Canada) by 6 × 10 cells per well. After a
were then subjected to a confocal laser scanning microscopy 24-h cultivation, the rBMSC microspheres were formed.
(LSM 880, Carl Zeiss, Germany) for fluorescence staining The microsphere size was measured using ImageJ
image observation. rBMSCs cultured in the chondrogenic software. SEM was employed to observe the microscopic
medium served as the positive control group. The morphology of rBMSC microspheres after lyophilization.
quantitative analysis of the relative percentage area coverage The rBMSC microspheres were subjected to staining of the
of ALP and fluorescence intensity of COL II were measured nucleus and F-actin. Briefly, the rBMSC microspheres were
and analyzed by ImageJ software. fixed with 4% PFA for 20 min, permeabilized with 0.2%
Triton X-100 for 20 min, and then incubated in 1% BSA
2.7. In vitro release behavior of scaffolds with for 1 h. Subsequently, samples were treated with a solution
different concentrations of growth factors of Alexa Fluor 594 phalloidin for the F-actin staining for 1
The in vitro release of the subchondral layer with BMP-2 in h at 37°C and with a solution of DAPI for nucleus staining
different amounts (1, 2, 3, and 4 μg BMP-2 per g scaffold) for 10 min at room temperature. Samples were subjected

Volume 10 Issue 5 (2024) 201 doi: 10.36922/ijb.3229