Nanofibrous biologic laminates replicate the form and function of the annulus fibrosus

Abstract

Successful engineering of load-bearing tissues requires recapitulation of their complex mechanical functions. Given the intimate relationship between function and form, biomimetic materials that replicate anatomic form are of great interest for tissue engineering applications. However, for complex tissues such as the annulus fibrosus, scaffolds have failed to capture their multi-scale structural hierarchy. Consequently, engineered tissues have yet to reach functional equivalence with their native counterparts. Here, we present a novel strategy for annulus fibrosus tissue engineering that replicates this hierarchy with anisotropic nanofibrous laminates seeded with mesenchymal stem cells. These scaffolds directed the deposition of an organized, collagen-rich extracellular matrix that mimicked the angle-ply, multi-lamellar architecture and achieved mechanical parity with native tissue after 10 weeks of in vitro culture. Furthermore, we identified a novel role for inter-lamellar shearing in reinforcing the tensile response of biologic laminates, a mechanism that has not previously been considered for these tissues.

Fig. 1. Fabrication of bi-lamellar tissue constructs

Scaffolds were excised 30° from the prevailing fiber direction of electrospun nanofibrous mats to replicate the oblique collagen orientation within a single lamella of the annulus fibrosus (a). At 0 weeks, MSC-seeded scaffolds were formed into bilayers between pieces of porous polypropylene and wrapped with a foil sleeve (b). Bilayers were oriented with either Parallel (+30°/+30°) or Opposing (+30°/-30°) fiber alignment relative to the long axis of the scaffold (c). P = porous polypropylene; F = foil; L1/2 = lamella 1/2. Scale = 25 μm (a).

Fig. 2. Elaboration of extracellular matrix within bilayers seeded with mesenchymal stem cells

Sulphated glycosaminoglycan (s-GAG, a) and collagen (b) content of Parallel and Opposing bilayers increased with culture duration (p ≤ 0.05). There were no significant differences between Parallel and Opposing bilayers at any time point. Alcian Blue (c), Picrosirius Red (d), and DAPI (e) staining of Opposing bilayer cross-sections after 10 weeks of in vitro culture. DW = dry weight. Dashed line indicates content at 0 weeks, when bilayers were formed. Error bars (a, b) represent the standard deviation of the mean. * indicates inter-lamellar space. Scale bar = 250 μm (c, d), 200 μm (e).

Fig. 3. Angle-ply collagen alignment and orientation

Sections were collected obliquely across lamellae (a), stained with Picrosirius Red, and viewed under polarized light microscopy to visualize collagen organization. When viewed under crossed polarizers, birefringent intensity indicates the degree of alignment of the specimen, while the hue of birefringence indicates the direction of alignment. After 10 weeks of in vitro culture, Parallel bilayers contained co-aligned intra-lamellar collagen within each lamella (b). Opposing bilayers contained intra-lamellar collagen aligned along two opposing directions (c), successfully replicating the gross fiber orientation of native bovine annulus fibrosus (d). In engineered bilayers, as well as the native annulus fibrosus, a thin layer of disorganized (nonbirefringent) collagen was observed at the lamellar interface (denoted by *). The distribution of collagen fiber orientations was determined by quantitative polarized light analysis. Prominent peaks in fiber alignment were observed near 30° in both lamellae of Parallel bilayers (e); however in Opposing bilayers two fiber populations were observed, aligned along +30° and -30° (f). Scale bar = 200 μm (b, c), 100 μm (d). L1/2 = Lamella 1/2; IL = Inter-lamellar space.

Fig. 4. Relating inter-lamellar mechanics with the tensile response of biologic laminates

Uniaxial tensile moduli of MSC-seeded Parallel and Opposing bilayers increased with in vitro culture duration, with Opposing bilayers achieving significantly higher moduli than Parallel bilayers from 4 weeks onward (a). # = p ≤ 0.05 compared to single lamellar modulus at 0 weeks. + = p ≤ 0.05 compared to Parallel modulus. Native = circumferential tensile modulus of native human AF². Lap testing of MSC-seeded laminates showed increasing interface strength with in vitro culture duration (b). # = p ≤ 0.05 compared to 2 weeks. To elucidate the role of interface properties on the tensile response of bilayers, uniaxial tensile testing was performed on acellular bilayers formed from nanofibrous scaffolds bonded together by agarose of increasing concentrations (c). Increasing inter-lamellar agarose concentration – and hence inter-lamellar stiffness – significantly increased the tensile modulus of acellular Opposing bilayers, but had no effect on the Parallel bilayer group. # = p ≤ 0.05 compared to orientation-matched 2% agarose. + = p ≤ 0.05 compared to concentration-matched Parallel bilayers. All error bars (a-c) represent standard deviations of the mean.

Fig. 5. A novel mechanism for tensile reinforcement by inter-lamellar shearing of biologic laminates

In Opposing bilayers (top), fibers within each lamella reorient under uniaxial load by rotating (red arrowheads) toward the loading direction. The opposing direction of rotation between lamellae generates shearing of the inter-lamellar matrix. However, in Parallel bilayers (bottom) fibers reorient identically in the two lamellae, and therefore do not shear the inter-lamellar space. L1/2 = Lamellae 1/2; IL = Inter-lamellar space.