Page 102 - IJB-6-3

3D printed gene-activated implants for bone regeneration
2 cm below and in parallel to the lower edge of
the mandible from its angle by 4 cm forward, soft
tissues were dissected, a surface of the mandibular
body exposed subperiosteally. Osteotomy was
performed by removing a lower edge fragment
sized 25 × 15 × 10 mm and retaining the mandible
3
continuity. 3D printed gene-activated implant was
fixed on the right side and plasmid-free 3D printed
implant (as control) fixed on the left one within
the defect using a straight titanium miniplate and
miniscrews (a diameter of 2 mm, a length of 5 –
7 mm). The post-operative wounds were closed in
four layers with Polysorb 4/0, SurgiPro 4/0.
CT: Having been fixed for 5 days, the materials
were examined with medical CT in the same
scanning mode and parameters, i.e. voxel size
0.08 mm, 80 kV, and 2 mA. The images were
analyzed with standard tools in the Planmeca
Romexis viewer software (Planmeca Oy, Finland).
A quantitative assay of the images involved the
determination of newly formed tissues density (in
Hounsfield units (HU)) in three projections with
apply of a region of interest tool and sizes of the
remaining implants.
SEM: The bone fragments lengthwise
through the central axis were cut. Several
samples sized 5 × 5 × 5 mm were resected from
3
each part of every material from the border
between the implant and a bone edge and from
an implant central zone for SEM study. The
remaining materials were used for a histologic
examination. SEM study was performed after
standard sampling with gold sputtering (section
2.3). Changes in the experimental samples’
structure, chemical compositions, and the border
between the implant and newly formed bone
were evaluated.
Histological analysis: 5-μm thick slices were
prepared from intact parts of every material
without previous decalcification and stained with
trichrome. The materials sampled for SEM were
decalcified in a Biodec-R solution and used for
histological specimens’ preparation under the
standard protocol with hematoxylin and eosin
staining. All specimens were scanned in a Mirax
Scanner (Carl Zeiss, Germany), and digital
images were generated and evaluated qualitatively
98 International Journal of Bioprinting (2020)–Volume 6, Issue 3
and quantitatively in the Panoramic Viewer
(3DHistech Ltd, USA).
2.6 Statistical analysis
All results are expressed as mean±SD. Mann–
Whitney U-test was used to compare independent
groups, with Wilcoxon signed-rank test used for
intra-group comparisons for each time points.
P <0.05 was considered statistically significant.
3 Results
All initial implants were produced by our
modified 3D printing technology [11,12] of ceramic
constructions from a synthesized TCP powder
and 1.0% aqueous solution of phosphoric acid
salt as described above. Experimental samples set
reproduced a shape, sizes and the structure of CAD
(computer assisted design) STL-models with a high
(ca. ± 100 µm) spatial accuracy and comprised:
1) non-porous disks to assess biodegradation; 2)
porous disks to evaluate a level of plasmid DNA
delivery; and 3) complex custom-made implants
to reconstruct the tibia and mandible large defects
in pigs.
3D printing fusing of TCP agglomerated
particles and “ink” (diluted phosphoric acid) is
based on a hydraulic setting reaction leading
to formation of dicalcium phosphate dihydrate
(DCPD), and thus, to layer-crossing bonding of the
powder. Finally, 3D structure is formed. According
to the XRD analysis, the 3D printed samples
compose of unreacted TCP and certain amounts of
DCPD (Figure 2A). FTIR and SEM data of the
experimental samples confirmed the formation
of DCPD (Figure 2B and C). SEM analysis of
the 3D printed samples are shown in Figure 2C.
Particles was about 5 – 20 μm in dimension. The
DCPD crystals had a flower-like morphology. The
width of the DCPD crystals was in the range of
1 – 50 μm, and their thickness was few microns.
Compressive strength of the 3D printed samples
is presented on Figure 2F and was in the range of
1.5 – 4.5 MPa, depending on the number of micro-
drops from printing head.
The post-treatment of the 3D printed samples
lead to formation of OCP phase and the adhesive