Engineered disc-like angle-ply structures for intervertebral disc replacement

Abstract

Study design: To develop a construction algorithm in which electrospun nanofibrous scaffolds are coupled with a biocompatible hydrogel to engineer a mesenchymal stem cell (MSC)-based disc replacement.

Objective: To engineer a disc-like angle-ply structure (DAPS) that replicates the multiscale architecture of the intervertebral disc.

Summary of background data: Successful engineering of a replacement for the intervertebral disc requires replication of its mechanical function and anatomic form. Despite many attempts to engineer a replacement for ailing and degenerated discs, no prior study has replicated the multiscale hierarchical architecture of the native disc, and very few have assessed the mechanical function of formed neo-tissues.

Methods: A new algorithm for the construction of a disc analogue was developed, using agarose to form a central nucleus pulposus (NP) and oriented electrospun nanofibrous scaffolds to form the anulus fibrosus region (AF). Bovine MSCs were seeded into both regions and biochemical, histologic, and mechanical maturation were evaluated with in vitro culture.

Results: We show that mechanical testing in compression and torsion, loading methods commonly used to assess disc mechanics, reveal equilibrium and time-dependent behaviors that are qualitatively similar to native tissue, although lesser in magnitude. Further, we demonstrate that cells seeded into both AF and NP regions adopt distinct morphologies that mirror those seen in native tissue, and that, in the AF region, this ordered community of cells deposit matrix that is organized in an angle-ply configuration. Finally, constructs demonstrate functional development with long-term in vitro culture.

Conclusion: These findings provide a new approach for disc tissue engineering that replicates multi-scale form and function of the intervertebral disc, providing a foundation from which to build a multi-scale, biologic, anatomically and hierarchically relevant composite disc analogue for eventual disc replacement.

Figure 1

(A) Schematic showing of fabrication process for formation of engineered disc-like angle-ply structure (DAPS) (NFS: nanofibrous scaffold). (B) Gross morphology of DAPS with nanofibrous AF region and agarose NP region, scale bar: 1 mm. (C) Close up view of AF region enlarged from box in (B). Representative stress-relaxation (D) and torsion (E) response of DAPS showing the viscoelastic and non-linear response of the composite.

Figure 2

(A) SEM of AF region after 1 week of culture. (B) Higher magnification SEM of interface formation between individual lamellae at 1 week time point. Actin (green) and DAPI (blue) staining of cells (C) and Picrosirius Red staining of newly formed collagen (D) organized in alternating directions along interface within sections taken oblique to the axial plane. Scale bar: 250 microns.

Figure 3

(A) DAPI staining of transverse section of DAPS at 1 week, showing homogenous distribution of MSCs in the ‘NP’ region, and lamellar organization of MSCs in the ‘AF’ region. Scale: 500 microns. Note: separation of NP and AF occurred as an artifact of sectioning. (B) Polarized light image of Picrosirius Red-stained oblique section of ‘AF’ region at 6 weeks showing birefringent material in opposing orientations with progression through adjacent lamellae. Scale: 100 (C) and 250 (D) microns.

Figure 4

Alcian Blue (A) and Picrosirius Red (B) staining of 6 week constructs with magnified images from NP and AF regions shown in (C) and (D) as indicated. Scale = 500 microns (A–B), 125 microns (C–D).

Figure 5

DNA (A), GAG (B), and collagen (C) content, reported in % wet weight (%WW) for the ‘NP’ and ‘AF’ regions as a function of time in culture. Solid and dashed lines indicate native lapine AF and NP benchmarks, respectively. ^#indicates p<0.05 for compared to time-matched ‘NP’ values; * indicates p<0.05 for compared to 1 week time point. Results presented as the mean ± SD for 4 samples/group per time point.

Figure 6

Equilibrium modulus (A) and percent stress relaxation (B) measured by unconfined compression for the DAPS as a function of time. ^# indicates p ≤ 0.05 compared to the 1 week time point. Results presented as the mean ± SD for 4 samples/group per time point.