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3D printed gene-activated implants for bone regeneration
disintegration and branching proximal and distal
cortical defects of the diaphyseal anterior surface
sized 10 × 5 × 5 mm each; a full-layer defect of
3
the mandible lower edge from the angle to the
front sized 25 × 15 × 10 mm . The specified zones
3
were segmented with the removal of surrounding
tissues. 3D reconstructions of designated bone
regions, corresponding to the planned bone defects
were transferred into MeshLab (Visual Computing
Lab, Italy), a mesh-object was generated,
transferred into Blender and the implant structure
was corrected, i.e. irregularities were removed,
perforating canals with a diameter of 1 mm were
added, a central opening with a diameter of 10 mm
created in the circular implant part corresponding
to the medullary canal for tibia reconstruction. The
resulting STL-format models were loaded into the
custom-made 3D printer software.
2.2 Post-treatment of 3D printed implants
3D printed samples were supplied for the post-
treatment into biomimetic solution, which was
produced by dissolving 115 g of monoammonium
phosphate (NH H PO ) in 500 mL of distilled water
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at room temperature and pH value 4.1 ± 0.1. 3D
printed samples were kept there at 40°C up to 168 h.
After that, the samples were thoroughly washed in
distilled water at least 10 times, dried in air at 37°C
and placed in a second solution, which was prepared
by dissolving 95.2 g of CH COONa in 700 mL of
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distilled water at 40°C and pH value 8.2 ± 0.2. 3D
printed samples were again kept at 40°C up to 168 h,
then washed in distilled water for at least 10 times
and dried in air at 37°C . All used reagents were
[11]
purchased from Sigma-Aldrich (USA).
2.3 Characterization of 3D printed implants
The porosity, intergranular size, and specific
surface area were studied by mercury porosimeter
(TriStar 3000, Micromeritics, USA). The phase
composition was analyzed by conventional X-ray
diffraction (XRD) (Shimadzu XRD-6000, Japan),
with Ni-filtered CuKα1 target, λ =1.54183 Å.
The samples were scanned from 2θ =3° to 60°
with a step size of 0.02° and a preset time of
5 s. Scanning electron microscope (SEM) (Tescan
96 International Journal of Bioprinting (2020)–Volume 6, Issue 3
Vega II, Czech Republic) equipped with EDS
analyzer, operating in secondary and backscattered
electron modes, was used for investigation of
surface morphology, microstructure, and chemical
composition. For SEM analysis, all samples were
sputter-coated before imaging with a 25 nm-thick
gold layer to impart electrical conductivity to the
specimen surfaces. Fourier transform infrared
(FTIR) spectroscopy study was performed using an
Infrared Spectroscopy microscope (Nicolet Avatar
330 FTIR spectrometer, UK) in transmission mode.
FTIR data were recorded over the range of 4000 –
−1
400 cm with 128 scans. The compressive strength
of the samples was evaluated in accordance with
the ISO standard 83.100: Cellular materials. At
least five samples for each experimental point were
tested. The compression test was carried out using
an Instron 5581 (Bucks, UK) testing machine
operating at a crosshead speed of 1 mm × min .
−1
2.4 Plasmid DNA deposition on 3D printed
implants
The supercoiled naked plasmid DNA
encoding VEGFA gene, an active substance
of “Neovasculgen,” the drug indicated for the
treatment of patients with chronic lower limb
ischemia (developed and certified for clinical
applications by PJSC Institute of Human Stem
Cells, Russia), was used to create personalized
gene-activated implants . Plasmid DNA carrying
[13]
the gene of luciferase (Luc) was also used in our
experiments to evaluate the transfection efficacy
in vivo. The combination of 3D printed bone
substitute with plasmid DNA was performed
under previously developed protocol . Briefly,
[14]
3D printed scaffolds were washed in a 0.5 M
solution of sodium phosphate monobasic dihydrate
(NaH PO × 2H O, Chimmed, Russia) at 37°С
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when constantly shaken for 10 h, washed in 1 ml of
10 mM NaH PO ×2H O at 37°С when constantly
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shaken 4 times for 10 min, then were left at 37°С
for 10 h until dried. After that, the samples were
placed in a 10 mM NaH PO ×2H O solution with
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plasmid DNA in the concentration of 1 μg/μl and
incubated at 37°С and constant shaking for 10 h,
and then a non-bound fraction of gene constructs
was washed with 5 mM NaH PO ×2H O.
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