Page 142 - IJB-9-3

International Journal of Bioprinting Bioprinting tissue-engineered bone-periosteum biphasic complex.

clarify the advantages and prospects of co-culture bioprinting 5’-GGAACAGGGTGGTGGAAGAC-3’ (reverse);
in the construction of complex living tissues and organs, and glyceraldehyde phosphate dehydrogenase (GAPDH) –
to promote the clinical translational research of composite 5’-AGACACGATGGTGAAGGTCG-3’ (forward),
biomaterials, stem cell differentiation, and bioprinting. 5’-TGCCGTGGGTGGAATCATAC-3’ (reverse). The qPCR
program was as follows: denaturation at 95°C for 10 s, and
2. Materials and methods 40 cycles at 95°C for 10 s, and 60°C for 30 s. The results
2.1. Cell culture and co-culture were analyzed using the comparison CT (2 − ΔΔCt ) method.
2.1.1. Culture of BMSCs and PDSCs of rabbit GAPDH was used as an internal control, and each sample
BMSCs of rabbit (rabBMSCs) were acquired according to was analyzed in triplicate.
our previous study . Briefly, bone marrow blood of New 2.1.4. Co-culture of rabBMSCs and rabPDSCs
[22]
Zealand White rabbit was extracted from the iliac bone, We co-cultured rabBMSCs and rabPDSCs using 0.4 μm
and then cultured in high-glucose Dulbecco’s modified polycarbonate membrane transwell insert (Corning, USA).
eagle’s medium (DMEM; Hyclone, USA) with 10% fetal In this experiment, three groups of cells were used: (I)
bovine serum (FBS; Gibco, USA), 100 U/mL penicillin, and rabBMSCs, (II) rabPDSCs, and (III) rabBMSCs+rabPDSCs.
100 μg/mL streptomycin (Hyclone, USA). PDSCs of rabbit In group (III), rabPDSCs were seeded onto the upper
(RabPDSCs) were obtained by enzymatic digestion as layer of the chamber. After culturing for 24 h, osteogenic
previously described [12,23] . In brief, the periosteum of rabbit induction medium was replaced, while the cells of the
skull was carefully harvested and digested by 0.25% type I control group were still cultured in complete DMEM.
collagenase (Sigma, USA). The digested suspension was then
filtered through a 70 μm nylon mesh, and then centrifuged 2.2. 3D extrusion-based bioprinting
at 1,500 rpm. The cells were resuspended in complete 2.2.1. Materials preparation
F-12 DMEM supplemented with 10% FBS, 100 U/mL The raw material of PLLA with molecular weights of
penicillin, and 100 μg/mL streptomycin for culturing. Cells 32,000 (3.2 W) and 54,000 (5.4 W) were purchased from
from the third to fifth generations were used. Daigang Inc (Shandong, China). HA (>97%, MW =
502.31) was purchased from Kingmorn Inc (Shanghai,
2.1.2. Multidirectional differentiation of rabBMSCs China). Gelatin of porcine skin was obtained from Sigma-
and rabPDSCs Aldrich (New Jersey, America). I-2959 (>98.0%, MW =
The primary cells were identified by muitidirectional 224.26), the photoinitiator, was purchased from TCI
differentiation. Alkaline phosphatase (ALP) staining was (Shanghai, China). Methacrylic anhydride (MA) and
used to analyze osteogenic differentiation after 7 days dichloromethane (≥99.5%, MW = 84.93) were purchased
of induction. Alizarin red S staining was used to detect from General Reagent of Titan Inc (Shanghai, China).
the mineralized nodules after 3 weeks. For adipogenic
differentiation, the newly produced lipid vacuoles could PLLA/HA composites with different ratios were
be confirmed by oil red O staining after 12 days. For prepared. Briefly, PLLA was dissolved in dichloromethane
chondrogenic differentiation, the cells that have been to form a transparent colloidal solution. HA powder was
induced for 3 weeks were detected by Alcian blue staining. then added to the PLLA solution at mass fraction of 10%,
20%, and 30%. The synthesis of GelMA has been reported
2.1.3. Real-time polymerase chain reaction (qPCR) previously . Briefly, gelatin was dissolved in phosphate-
[24]
Total RNA was extracted form rabBMSCs and rabPSDCs buffered saline (PBS, 10% (w/v)), and then MA was added
using Trizol reagent (Invitrogen, USA) after 7 days of (20% v/v). The fully mixed solution was then reacted at
osteogenic differentiation. After the reverse transcription 50°C for 3 h, and then dialyzed with distilled water for
reaction, real-time polymerase chain reaction (qPCR) 1 week. Finally, the GelMA precursor could be obtained by
was performed with a QuantStudio 6Flex system (Life freeze-drying and stored for further use.
Technologies, USA) using SYBR Premix (Takara, Japan). The The precursor of GelMA and I-2959 were dissolved
primer sequences used in this study were described as follows: in PBS (5% (w/v) hydrogel solution with 0.1% (w/v)
collagen I (COL1) – 5’-CAGCGGCTCCCCATTTTCTA-3’ photoinitiator). Next, rabBMSCs and rabPDSCs were
(forward), 5’-ATCTCAGCTCGCATAGCACC-3’ (reverse); mixed with the prepared hydrogel at a density of 5 × 10 /mL.
6
osteocalcin (OCN) – 5’-AGAGTCTGGCAGAGGCTCA-3’ The prepared bioink was rewarmed before being added to
(forward), 5’-CAGGGGATCCGGGTAAGGA-3’ (reverse); the printing cartridges.
osteopontin (OPN) – 5’-AGCGTGGAAACCCAAAGTCA-3’
(forward), 5’-GCTCGATGGCTAGCTTGTCT-3’ (reverse); 2.2.2. 3D bioprinting process
runt-related transcription factor 2 (RUNX2) – The 3D Bioplotter printer (Envision Tec, Germany) was
5’-GATGACGTCCCCGTCCATTC-3’ (forward), applied to construct the complex structure. One of the

Volume 9 Issue 3 (2023) 134 https://doi.org/10.18063/ijb.698