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CTP Scaffolds Treated Bone Defects
cylindrical scaffolds were drawn (each layer consists of was added with 0.1 mM dexamethasone (Sigma-Aldrich,
seven parallel struts, and struts in adjacent layers had a St. Louis, MO. USA), 100 μg/mL ascorbic acid (Sigma-
cross angle of 90°, Figure S1-A). The printing process Aldrich), and 10 mM β-glycerol phosphate (Sigma-
is shown in Figure S1-B. The emulsion mixture was Aldrich). RAW264.7 cell line (ATCC) was maintained in
extruded by pushing the plunger of the syringe via an DMEM containing 10% FBS. The medium was changed
electromechanical screw, and the as-printed pattern every 2 days. For osteoclastic differentiation, cells were
was frozen in 2 s due to the existence of the cryogenic incubated with medium containing 50 ng/ml RANKL (R
environment (−30°C). The printing speed is 6 mm/s and and D Systems, Minneapolis, MN, USA) for 7 days. In the
the feeding rate of the plunger was set as 0.008 mm/s. control group and CFZ group, the cells were stimulated
After the cryogenic 3D printing, the as-fabricated scaffolds with dimethylsulfoxide (DMSO) and the extracts of
were freeze-dried to remove water and DCM. Thus, a scaffolds, respectively.
stable cylindrical bone tissue engineering scaffold with a
diameter of 3 – 4 mm and a height of 15 mm was obtained. 2.6. Western blot analysis
2.2. Morphology observation and structural The cells were lysed in 2% sodium dodecyl sulfate (SDS),
characterization analysis 2 M urea, 10% glycerol, 10 mM Tris-HCl (pH 6.8), 10 mM
dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride.
The surface morphology was observed by scanning Proteins were separated by 10% SDS-polyacrylamide gel
electron microscopy (SEM) (Gemini, Zeiss, Germany). electrophoresis. After electrophoresis, the proteins were
Before SEM, a thin layer of gold was sprayed on transferred to the membrane by wet transfer (Bio-Rad
the surface of the material to improve its electrical Laboratories, Hercules, CA, USA). Each membrane was
conductivity. The average pore size of the specimen was incubated with TBST (100 mM Tris-HCl pH 7.5, 150 mM
measured by SEM. On the basis of TP material, TCP/ NaCl, 0.05% Tween 20) and 5% non-fat blocking milk
PLGA slow-release CFZ was prepared by cryogenic 3D powder at room temperature for 1 h, and then incubated
printing to make CTP material. Micro-CT and SEM were overnight with the primary antibody in a shaking bottle
used to evaluate the structure of the CTP. at 4°C. The membrane and HRB-conjugated secondary
antibody were incubated at room temperature for
2.3. Mechanical properties of the scaffold 1 h. The membrane was then treated with enhanced
The scaffolds were 15 mm thick, 3.4 mm wide, and chemiluminescence reagent (ECL Kit, Amersham
2.3 mm high. TP and CTP materials were tested by an Biosciences, Piscataway, NJ, USA), and the proteins
electronic universal testing machine (SUNS, Shenzhen, were detected using chemiluminescence technology.
China). The test and load-displacement curve were 2.7. Gene expression and real-time polymerase
carried out at a speed of 1 mm/min and 40 s. Finally,
the corresponding values of compressive strength, chain reaction (PCR) analysis
compression modulus, and compressive strain were The expression of osteogenesis genes, such as Alkaline
obtained according to the GB/T 1041-92 calculation Phosphatase (ALP), bone morphogenetic protein 2 (BMP2),
standard. collagen type I, Osteocalcin (OCN), transcription factor SP7
(Osterix), and Runt-related transcription factor 2 (Runx2) in
2.4. In vitro release of the scaffold C3H10T1/2 cells, and of osteoclastogenic genes, such as

To study CFZ release in vitro, preweighed stent samples Cathepsin K (CTSK), Matrix metalloproteinase-9 (MMP9),
were placed in tubes containing phosphate-buffered c FBJ osteosarcoma oncogene (c-fos), Nuclear factor of
saline (PBS) solution (0.02% sodium azide for CFZ test). activated T cells cytoplasmic 1 (NFATC1) in Raw264.7 cells
The test tube was placed in an oscillating water bath at cultured in different treatments was detected by PCR. Total
37°C, the test solution was taken out at a predetermined RNA was extracted from the cells with Trizol reagent (Life
interval, and the concentration of CFZ was determined Technologies, Carlsbad, CA, USA). The concentration of
using the CFZ Determination Kit (US Biological, USA) RNA was determined by a NanoDrop spectrophotometer
and the release time was determined. (Thermo Fisher Scientific, USA). Primers for real time
PCR (RT-PCR) are listed in Table 1.
2.5. Cell culture
Mouse mesenchymal stem cells C3H10T1/2 were cultured 2.8. Alizarin red S staining
in Dulbecco’s Modified Eagle Medium (DMEM) medium C3H10T1/2 cells were seeded into 48-well plates (three wells
supplemented with 10% fetal bovine serum (FBS), 100 per group). After osteogenic induction for 14 days, the cells
U/mL penicillin G, and 100 mg/mL streptomycin under were fixed in 4% paraformaldehyde, and then rinsed twice
standard conditions. To induce osteogenesis, this medium in PBS. Afterwards, they were stained at room temperature

102 International Journal of Bioprinting (2021)–Volume 7, Issue 4