3D bioprinting of an intervertebral disc tissue analogue with a highly aligned annulus fibrosus via suspended layer additive manufacture

Abstract

Intervertebral disc (IVD) function is achieved through integration of its two component regions: the nucleus pulposus (NP) and the annulus fibrosus (AF). The NP is soft (0.3-5 kPa), gelatinous and populated by spherical NP cells in a polysaccharide-rich extracellular matrix (ECM). The AF is much stiffer (∼100 kPa) and contains layers of elongated AF cells in an aligned, fibrous ECM. Degeneration of the disc is a common problem with age being a major risk factor. Progression of IVD degeneration leads to chronic pain and can result in permanent disability. The development of therapeutic solutions for IVD degeneration is impaired by a lack ofin vitromodels of the disc that are capable of replicating the fundamental structure and biology of the tissue. This study aims to investigate if a newly developed suspended hydrogel bioprinting system (termed SLAM) could be employed to fabricate IVD analogues with integrated structural and compositional features similar to native tissue. Bioprinted IVD analogues were fabricated to recapitulate structural, morphological and biological components present in the native tissue. The constructs replicated key structural components of native tissue with the presence of a central, polysaccharide-rich NP surrounded by organised, aligned collagen fibres in the AF. Cell tracking, actin and matrix staining demonstrated that embedded NP and AF cells exhibited morphologies and phenotypes analogous to what is observedin vivowith elongated, aligned AF cells and spherical NP cells that deposited HA into the surrounding environment. Critically, it was also observed that the NP and AF regions contained a defined cellular and material interface and segregated regions of the two cell types, thus mimicking the highly regulated structure of the IVD.

Keywords: 3D bioprinting; annulus fibrosus; intervertebral disc; mesenchymal stem cell; nucleus pulposus.

Figure 1.

Schematic representation of the IVD bioprinting pipeline. (A) Design of an appropriate computer aided design (CAD) model. (B) SLAM bioprinting of an IVD analogue. (C) An example of a bioprinted analogue with a polysaccharide-rich core and collagenous, aligned AF. (D) Printing of a cell-laden IVD construct with embedded IVD cells or MSCs.

Figure 2.

Mechanical and structural characterisation of SLAM bioprinted IVD analogues. (A) Photographic image of a printed construct showing clearly defined NP and AF regions. (B) DMT modulus of the NP and AF regions at days 1 and 28 post-printing. N = 3; ****P < 0.0001; one-way ANOVA. (C) SEM, TEM and AFM of the NP and AF regions. (D) AFM and TEM of the NP-AF interface.

Figure 3.

Cell viability and tracking of IVD cells in bioprinted constructs. (A) Representative Live/Dead stain of human NP and AF cells within a bioprinted construct 24 h post-printing. (B) Whole bioprinted IVD showing partial overlay of printed cells labelled with cell tracker dyes. NP cells pseudo-coloured yellow, AF cells pseudo-coloured purple. Higher magnification images of NP and AF cells within each region and a combination of the two cell types with distinct morphologies at the interface. Staining performed on the constructs 28 d post-printing.

Figure 4.

Biological characterisation of SLAM bioprinted IVD analogues. (A) Representative actin and hyaluronan (HA) staining of NP and AF cells in a bioprinted construct 28 d post-printing showing regional morphology differences and hyaluronan expression by NP cells. Representative alcian blue-picrosirius red histology demonstrating integration of NP cell and AF cell-seeded NP and AF regions 28 d post-printing. (B) Representative actin and hyaluronan (HA) staining of MSCs in a bioprinted construct 28 d post-printing showing regional morphology differences and hyaluronan expression by NP cells. Representative alcian blue-picrosirius red histology demonstrating integration of MSC-seeded NP and AF regions 28 d post-printing.

Figure 5.

Alignment of collagen in the AF of SLAM bioprinted IVD analogues. AFM and pseudo-coloured AFM images, created using OrientationJ within ImageJ, demonstrating collagen alignment around the AF circumference. No orientation could be identified within the NP region (white dot).

Figure 6.

Collagen and cell alignment is triggered via bioprinting. (A)–(C) Pseudo-coloured images and corresponding coherency analysis for collagen in bioprinted IVD constructs and unprinted collagen gels. (D)–(F) Pseudo-coloured images and corresponding coherency analyses for AF cells in bioprinted IVD constructs and unprinted collagen gels. (G)–(I) Pseudo-coloured images and corresponding coherency analyses for MSCs in bioprinted IVD constructs and unprinted collagen gels. N = 3; ****P < 0.0001; one-way ANOVA. A.U. = arbitrary units.