In situ printing of mesenchymal stromal cells, by laser-assisted bioprinting, for in vivo bone regeneration applications

Abstract

Bioprinting has emerged as a novel technological approach with the potential to address unsolved questions in the field of tissue engineering. We have recently shown that Laser Assisted Bioprinting (LAB), due to its unprecedented cell printing resolution and precision, is an attractive tool for the in situ printing of a bone substitute. Here, we show that LAB can be used for the in situ printing of mesenchymal stromal cells, associated with collagen and nano-hydroxyapatite, in order to favor bone regeneration, in a calvaria defect model in mice. Also, by testing different cell printing geometries, we show that different cellular arrangements impact on bone tissue regeneration. This work opens new avenues on the development of novel strategies, using in situ bioprinting, for the building of tissues, from the ground up.

Figure 1

Schematic representation of the laser assisted bioprinting (LAB) approach. A typical LAB setup comprises a pulsed laser beam, a focusing system, a ribbon (a transparent glass slide, coated with a laser-absorbing layer of metal, onto which a thin layer of bioink is spread, and a receiving substrate facing the ribbon. The physical principle of LAB is based on the generation of a cavitation-like bubble, into the depth of the bioink film, whose expansion and collapse induces the formation of a jet and, thereby, the transfer of the bioink from the ribbon to the substrate (here a bone defect on the mouse calvaria), forming a microdroplet.

Figure 2

(A) Representative fluorescence images of ring and disk printed tomato-positive D1 cells at days 0, 2 and 4. (B) Percent metabolic activity, as measured by the resazurin assay, of D1 cells printed in a ring or disk geometry at days 1 and 8, in relation to ring geometry at day 1 (Average ± SD, n = 6, **and ***denotes p < 0.01 and p < 0.001, respectively).

Figure 3

Schematic representation of the in vivo laser assisted bioprinting geometries tested, namely a ring (A1) with external and internal diameter of 3 and 2.1 mm, respectively, and a disk (B1) with 2 mm diameter. In both cases, two layers of nHA-collagen ink were printed underneath and over the cellularized ink layer. Representative fluorescence images of ring (A2) and disk (B2) printed tomato-positive (D1) cells inside the calvaria defect in mice, immediately after printing.

Figure 4

(A) Representative luminescence imaging of luciferase positive D1 cells in a ring geometry at 10, 15, 21, 28, 35 and 42 days post printing, in a mice calvaria model. (B) Quantification of the luciferase signal of luciferase positive D1 cells in a ring and disk geometry in a mice calvaria model (Average ± SD, n = 5).

Figure 5

(A) Representative X-ray micro tomography (μCT) reconstruction images of nHA collagen and D1 cells printed in a ring or disk geometry (calvaria defect in the right side), or nHA collagen alone (calvaria defect in the left side), at 2 months post printing in a mice calvaria model. Horizontal (B) and coronal (C) μCT projection and regions of interest (3.3 mm diameter and 0.5 mm thick disk) for the evaluation of bone repair, in a calvaria defect in mice at 2 months post impression. (D) Quantitative assessment of bone volume/total volume (BV/TV) by μCT evaluation of nHA collagen and D1 cells, printed in a ring or disk geometry, or nHA collagen alone at 1 and 2 months post printing (Average ± SD, n = 9, *and ***denote p < 0.05 and p < 0.001, respectively).

Figure 6

Horizontal (A) and coronal (B) X-ray micro tomography (μCT) projection and region of interest (1.5 mm diameter and 0.5 mm thick disk) for the evaluation of bone repair, in the center of a calvaria defect in mice at 2 months post impression. (C) Quantitative assessment of bone volume/total volume (BV/TV) by μCT evaluation of nHA collagen and D1 cells, printed in a ring or disk geometry, or nHA collagen alone at 2 months post printing (Average ± SD, n = 9, *denotes p < 0.05).

Figure 7

Histologic evaluation by Hematoxylin/Eosin/Safran (HES) staining of bone repair, in a calvaria defect in mice, at 1 and 2 months post impression of nHA collagen and D1 cells, printed in a ring or disk geometry, or nHA collagen alone. (Representative images are shown).