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Zhang, et al.
solution of Triton X-100 in a protease inhibitor cocktail suspension cells after 48 h, and the adherent cells were
(0.1% w/v EDTA, 3.5% w/v PMSF in Tris-HCl, pH = 7.5) expanded up to passage 3. To encapsulate cells for 3D
for 24 h. Next, the cartilage and bone granules were bioprinting, BMSCs were trypsinized after reaching
washed and digested with 50 U/mL deoxyribonuclease approximately 80% confluence and then washed with
and 1 U/mL ribonuclease (Sigma-Aldrich, St. Louis, MO, low-glucose DMEM containing 10% fetal bovine serum.
USA) for 12 h. To obtain DCM and DBM, the granules After centrifugation at 1200 rpm for 5 min, BMSCs were
were lyophilized and solubilized using a previously resuspended in bioinks at a density of 1.0 × 10 cells mL .
−1
7
reported protocol with modifications . In brief, 10 mg
[30]
decellularized granules were mixed with 1 mL of 0.1 mol 2.5. Fabrication of bilayered scaffolds
HCl containing 1 mg of pepsin powder (Sigma-Aldrich, 3D bioprinting system (3D Discovery, Regenhu, Villaz-
St. Louis, MO, USA) at room temperature for 2 days. St-Pierre, Switzerland) provided by Bioexcellence Inc.
After solubilization, 1 Mol NaOH was added to adjust the (Beijing, China) was used to fabricate the bilayered
pH to 7.4. The solution was centrifuged at 10,000 ×g for scaffolds. DBM bioink and DCM bioink were prepared
3 min to remove undigested particles and the supernatant and loaded into 10 mL plastic containers at room
was lyophilized and stored at −80℃ for longer storage. temperature. FDA-approved PCL (molecular weight 70–
2.2. Preparation of TGF-β1-loaded DCM/SF 90 Kda) provided by Polysciences Inc. (PA, USA) was
bioink loaded into metal container with temperature control, and
the container temperature was set to 60°C. The parameters
Solubilized and lyophilized SF protein (SF, molecular related to the process of printing are listed in Table 1.
weight >100 kDa) was purchased from Simatech For the fabrication of the bone layer (4 mm in
Inc. (Suzhou, China). Polyethylene glycol (PEG 400; diameter, 4.5 mm in height), PCL was first extruded to
molecular weight 380~420 Da) was provided by Aladdin print outline, and then, the DBM/SF bioink was printed
Inc. (Shanghai, China). About 10% w/v of SF, 5% w/v to fill the space. DCM/SF bioink was used to print the
DCM, and 4 μg/mL of TGF-β1 (PeproTech, Rocky Hill, cartilage layer (4 mm in diameter, 0.5 mm in height) on
USA) were dissolved in PBS to prepare SF/DCM blends. the bone layer (Figure 1). The thicknesses of cartilage
The blends were then mixed with an equal volume layer and bone layer were adjusted to 2 mm for in vitro
of 80% v/v PEG for gelation of the DCM/SF bioinks investigation.
(5% w/v final SF concentration; 2.5% w/v final DCM
concentration; and 2 μg/mL final TGF-β1 concentration). 2.6. Fourier-transform infrared (FTIR)
spectroscopy
2.3. Preparation of BMP-2-loaded DBM/SF
The infrared spectra of DCM/SF and DBM/SF
bioink hydrogels were evaluated using Thermo Scientific
About 10% w/v of SF, 5% w/v DBM, and 4 μg/mL of Nicolet iS5 FT-IR Microscope (Waltham, MA, USA).
BMP-2 (PeproTech, Rocky Hill, USA) were dissolved in The samples were prepared by lyophilization for FTIR
PBS to prepare SF/DBM blends. The blends were then analysis. Scanning was conducted in the spectral range
mixed with an equal volume of 80% PEG for gelation from 1000 cm to 2200 cm .
−1
−1
of the DBM/SF bioinks (5% w/v final SF concentration;
2.5% w/v final DBM concentration; and 2 μg/mL final Table 1. Parameters of the designed construct and 3D bioprinting.
BMP-2 concentration). Parameters Cartilage layer Bone layer
2.4. Cell isolation and encapsulation DCM/SF DBM/SF PCL
bioink bioink
BMSCs were harvested and isolated as we previously Container 15°C 15°C 60°C
described [45,46] . Briefly, bone marrow aspirate was temperature
isolated from the medullary cavity of femur bone in New
Zealand rabbits. The mixture of cells was separated by Nozzle diameter 0.25 mm 0.25 mm 0.25 mm
gradient density centrifugation in 1.073 g/ml lymphocyte Size (length * 4 mm* 4 mm*4 mm*4.5 mm
separation solution (Gibco, NY, USA). The mononuclear width * height) 4 mm*0.5 mm
fraction interphase was collected and washed twice in Interlayer spacing 0.25 mm 0.25 mm 0.25 mm
sterile PBS. The cells were resuspended in low-glucose Line spacing 0.20 mm 0.20 mm 2 mm
DMEM containing 100 U/ml penicillin, 100 U/ml Printing speed 4 – 7 mm/s 3 – 1.8~2.7
streptomycin, and 10% fetal bovine serum (HyClone, 5 mm/s mm/s
UT, USA), and subsequently incubated at 37°C with Print pressure 0.20–0.30 MPa 0.20– 0.50~0.60
5% CO . The culture medium was changed to remove 0.30 MPa MPa
2
International Journal of Bioprinting (2021)–Volume 7, Issue 4 87