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. 2023 Mar 31;12(4):536.
doi: 10.3390/biology12040536.

Tackling Inequalities in Oral Health: Bone Augmentation in Dental Surgery through the 3D Printing of Poly(ε-caprolactone) Combined with 20% Tricalcium Phosphate

Nicola De Angelis  1   2   3 , Andrea Amaroli  4 , Maria Giovanna Sabbieti  5 , Alessia Cappelli  5 , Alberto Lagazzo  2 , Claudio Pasquale  1   2 , Fabrizio Barberis  2 , Dimitrios Agas  5
Affiliations

Tackling Inequalities in Oral Health: Bone Augmentation in Dental Surgery through the 3D Printing of Poly(ε-caprolactone) Combined with 20% Tricalcium Phosphate

Nicola De Angelis et al. Biology (Basel). .

Abstract

The concept of personalized medicine and overcoming healthcare inequalities have become extremely popular in recent decades. Polymers can support cost reductions, the simplicity of customized printing processes, and possible future wide-scale expansion. Polymers with β-tricalcium phosphate (TCP) are well known for their synergy with oral tissues and their ability to induce osteoconductivity. However, poor information exists concerning their properties after the printing process and whether they can maintain an unaffected biological role. Poly(ε-caprolactone) (PCL) polymer and PCL compounded with TCP 20% composite were printed with a Prusa Mini-LCD-®3D printer. Samples were sterilised by immersion in a 2% peracetic acid solution. Sample analyses were performed using infrared-spectroscopy and statical mechanical tests. Biocompatibility tests, such as cell adhesion on the substrate, evaluations of the metabolic activity of viable cells on substrates, and F-actin labelling, followed by FilaQuant-Software were performed using a MC3T3-E1 pre-osteoblasts line. PCL+β-TCP-20% composite is satisfactory for commercial 3D printing and appears suitable to sustain an ISO14937:200937 sterilization procedure. In addition, the proper actin cytoskeleton rearrangement clearly shows their biocompatibility as well as their ability to favour osteoblast adhesion, which is a pivotal condition for cell proliferation and differentiation.

Keywords: additive manufacturing; bone augmentation; bone defect; dentition; oral disorder; polymer; rapid prototyping; socket.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The poly(ε-caprolactone) (PCL) polymer and the PCL compounded with beta-tricalcium phosphate 20% (β-TCP 20%) composite polymer was printed according to the experimental setup shown in (A) and sterilised with a 2% peracetic acid solution. The PCL and β-TCP 20% printed samples were photochemically (B) and mechanically (C) characterised. The biocompatibility of the β-TCP 20% samples (D) was tested to show the effects of their interaction with preosteoblast cells. For this, the ability of the cells to adhere to the substrate (a), their cytoskeletal re-organisation (b), and the activity of cell metabolism (c) were assayed.
Figure 2
Figure 2
Description of static mechanical tests. The samples were placed on two support anvils and bent through an applied force on one or two anvils to measure their stress–strain data. The beam was proportioned so that it did not fail through shear or by lateral deflection before reaching its ultimate flexural limit. The equation reported for the highest stress at the moment of rupture was employed, where F = the load at the bar centre, L = the distance between the two lower supports, w = the width of the specimen, and h = the thickness of the specimen.
Figure 3
Figure 3
(A) Fourier transform infrared (FT-IR) spectrum of poly(ε-caprolactone) (PCL) samples. The black arrow indicates that there was no interaction in the ester group between the unsterilized PCL (blue line) and sterilized PCL (red line) samples. (B) FT-IR spectrum of PCL compounded with beta-tricalcium phosphate 20% (PCL+β-TCP 20%) samples. The green arrows indicate the interactions in the ester group between the unsterilized composite (blue line) and the sterilized composite (red line) samples.
Figure 4
Figure 4
Graphical representations of the five analyses of unsterilised (A) and sterilised (B) poly(ε-caprolactone) (PCL) samples. No differences in deformation (%) were noticed in the range of observation.
Figure 5
Figure 5
Graphical representations of the five analyses of unsterilized (A) and sterilized (B) poly(ε-caprolactone) (PCL) compounded with beta-tricalcium phosphate 20% (PCL+β-TCP 20%) samples. No differences in deformation (%) were noticed in the range of observation.
Figure 6
Figure 6
(A) Spatial representation of the poly(ε-caprolactone) (PCL) compounded with beta-tricalcium phosphate 20% (PCL+β-TCP 20%)-based biomaterial with toluidine blue-stained preosteoblasts, magnification 20×. (B) Surface plot of the PCL+β-TCP 20% compounds with the relevant expansion of white dots (pre-osteoblasts) on almost the whole surface. (C) Toluidine blue staining of preosteoblasts expanding on the central meshes or the peripheral (D,E) meshes of the compound, magnification 20× (C,D) and 40× (E, as magnification of black dashed square of D). The hematoxylin/eosin staining of the proliferating preosteoblasts on the biomaterial network better elucidates the affinity of the compound with the developing cells in the centre (F) or peripheral parts of the substrate (G).
Figure 7
Figure 7
MTS assay. Note the suitability of the poly(ε-caprolactone) (PCL) compounded with beta-tricalcium phosphate 20% (PCL+β-TCP 20%) samples for supporting the growth and viability of the preosteoblasts relative to those cultured in the normal culture dishes. The values from three different experiments were calculated as the means ± the standard deviations. No statistically significant differences were observed (p > 0.05).
Figure 8
Figure 8
Preosteoblast growth over the poly(ε-caprolactone) (PCL) compounded with beta-tricalcium phosphate 20% (PCL+β-TCP 20%)-based substrate which was stained with phalloidin (TRITC-conjugate) for F-actin detection. (A) Map of the minimum of n nearest neighbour cells on the compound. (B) Confocal microscopy captured the preosteoblast actin filaments spreading on the surface of the substrate. (C–H) Confocal microscopy 3D illustration of the phalloidin-labelled F-actin of preosteoblasts over the substrate. Note the F-actin distribution over the substrate in multiple scans. (I,J) 3D surface plot of the substrate. (K) Projections of cell maps on the biomaterial. The numbers in yellow represent the pixel distances between the cultured cells and the substrate. (L) A map of proximal neighbour preosteoblasts on the substrate meshes. Note the actin adhesion points (white arrows) between the cells (M) and the actin filament skeleton in expansion on the substrate (N). (O) Filopodium formation is tagged with a purple colour. The open-source image processing software ImageJ [version ImageJ2 2.9.0/1.53t] was used for image analysis [47].
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