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The binding of plasmid DNA with OCP surface implants with VEGFA-carrying (Control 2) or
was controlled visually by SEM and chemically Luc-carrying plasmid DNA (test group) were
when a bound plasmid DNA fraction was taken off inserted subcutaneously to an animal. Shame-
with 0.5 M NaH PO ×2H O, and its concentration operated animals (Control 3) and those injected
4
2
2
in a solution was measured with fluorometer Qubit with a solution of plasmid DNA with Luc gene
2.0 (Invitrogen, USA). (Control 4) were used as additional controls. On
days 1, 7, 14, and 28, D-luciferin sodium salt
2.5 In vivo experiments (Lumtec, Russia) was intraperitoneally injected
Animal trials were carried out in accordance with to animals which had been preliminarily sedated
institutional guidelines/protocols in agreement with Sol. Zoletili 100 – 10 mg/kg. In 10 min,
with national laws and policies for animal care. they were placed in a bioluminometer IVIS
The guidelines were approved by PJSC Institute Spectrum chamber (PerkinElmer, Inc., USA), and
of Human Stem Cells, Moscow, Russia. a luminescent signal was recorded for 1 min.
2.5.1 Biodegradation assessment 2.5.3 Segmental bone defect reconstruction
Male pigs with an average body weight of
Biodegradation assessment of 3D printed OCP 50 ± 2 kg (n = 4) were used in this study. Each
based and gene-activated implants were studied in animal underwent surgery on the right tibia and
male rats with a body weight of 150 g (n = 24). the mandible on both sides under combined
A 15-mm median skin incision was done in the endotracheal anesthesia. After the surgery, all
lower back under inflatration anesthesia with Sol. animals received antibiotic therapy (Cefazolin-
Lidocaini 1 – 2 ml and intramuscular sedation natrii 1.0) for 7 days and then were sacrificed by
with Sol. Zoletili 100 – 10 mg/kg and 20 mm thiopental sodium overdosage when sedated with
subcutaneous pockets were formed on both Sol. Zoletili 100 in 3- and 6-months post-surgery.
sides of the incision. The gene-activated bone The bones with the previous surgery areas were
substitutes were implanted into the right zone, but resected. Reconstructive plates and screws were
3D printed scaffolds without plasmid DNA – into removed from the tibia; the metal constructions
the left one. A post-operative wound was sutured were left in the mandible to retain the regeneration
by interrupted stitches with Polysorb 5/0. The integrity. The materials were fixed in 10% neutral
animals were sacrificed in 15, 30, 90, 120, and formalin.
180 days by overdosage of Sol. Zoletili 100. The Tibia reconstruction: 10-cm straight incision
materials were extracted and fixed in 10% neutral was made through the skin along the anterior tibia
formalin. Micro CT of the samples was performed surface, soft tissues were dissected, the diaphysis
by Brucker SKYSCAN 1174 (Belgium) and then exposed subperiosteally and a T-shaped fragment
the images were 3D-reconstructred using VG corresponding to a planned bone defect with a total
Studio Max software (Germany). A diameter, length of 30 mm was resected. 3D printed gene-
shape, and a surface area to implant volume activated implant was inserted into the defect, and
ratio were determined. After decalcification in a osteosynthesis performed with the use of a 3.2
Biodec-R solution (Bio-optica, Italy), histological mm-thick reconstructive plate and screws with
slices were prepared under a standard procedure a diameter of 3.7 mm, some of them were fixed
followed by hematoxylin and eosin staining. bicortically. Bone fragments were fixed stable on
2.5.2 Gene constructs delivery assessment a surgical table. The post-operative wounds were
in vivo closed in four layers with Polysorb 4/0, SurgiPro
4/0.
Balb/c mice with a body weight of 30 g (n = 20) Mandible reconstruction: the intervention was
were used in this experiment. 3D printed carried out on both sides of the mandible under
scaffolds (Control 1), 3D printed gene-activated the same protocol. A skin linear incision was made
International Journal of Bioprinting (2020)–Volume 6, Issue 3 97
