Replication of an intervertebral disc using an in-house bioprinter: A proof-of-concept study

Abstract

The intervertebral disc, a fibro-cartilaginous structure, contains a gelatinous core (nucleus pulposus) surrounded by collagen fiber lamellae (annulus fibrosus). Intervertebral disc degeneration leads to a debilitating disorder, and new treatments are currently being developed. Unfortunately, conventional monolayer in vitro models fail to predict the clinical efficacy of novel therapies accurately. Here, we report a bioprinted construct that mimics the macroscopic and microscopic architecture of the intervertebral disc. First, a 3D model was created from a histological section of a sheep lumbar intervertebral disc. The printability of the ink, gelatin (7% w/v), alginate (0.6% w/v), and hyaluronic acid (0.2% w/v) was optimized by varying the printing pressure (70–110 kPa), printing speed (2–10 mm/s), and nozzle type (needle or tip). Nucleus pulposus and annulus fibrosus cells, harvested from 4-month-old lambs, were bioprinted (5 × 105 cells/mL) using an in-house extrusion bioprinter. Cell viability (live/ dead assay), shape (actin immunostaining), distribution (confocal microscopy), and matrix synthesis (immunostaining) were evaluated after 21 days of culture. We used a parametric study to quantify and optimize the factors (pressure, printing speed, nozzle type) influencing the filament width. The 3D construct exhibited fidelity to the initial design and maintained stability in length, width, and height for 21 days. Fluorescent labeling confirmed the distribution of nucleus pulposus and annulus fibrosus cells in each tissue, replicating the native intervertebral disc structure. We also evidenced cell viability and collagen type 1 synthesis. This bioprinted construct offers a promising alternative to current in vitro models, potentially enabling more relevant preclinical evaluations.

Keywords

3D bioprinting / Alginate / Hydrogel / In-house extrusion bioprinter / In vitro 3D model / Gelatin / Hyaluronic acid

Funding

The authors acknowledge the SC3M platform from the INSERM/NU/ONIRIS UMR1229 RMeS Laboratory (SFR Bonamy, BioCore, Inserm UMS 016, CNRS UAR 3556, Nantes, France) and the IBISA MicroPICell facility (Biogenouest, France), a member of the national infrastructure France-Bioimaging, supported by the French National Research Agency (ANR-10-INBS-04). The authors thank Hilel Moussi for their assistance. The graphical abstract was created using BioRender.

Conflict of interest

The authors declare they have no competing interests.