Intervertebral disk tissue engineering using biphasic silk composite scaffolds

Abstract

Scaffolds composed of synthetic, natural, and hybrid materials have been investigated as options to restore intervertebral disk (IVD) tissue function. These systems fall short of the lamellar features of the native annulus fibrosus (AF) tissue or focus only on the nucleus pulposus (NP) tissue. However, successful regeneration of the entire IVD requires a combination approach to restore functions of both the AF and NP. To address this need, a biphasic biomaterial structure was generated by using silk protein for the AF and fibrin/hyaluronic acid (HA) gels for the NP. Two cell types, porcine AF cells and chondrocytes, were utilized. For the AF tissue, two types of scaffold morphologies, lamellar and porous, were studied with the porous system serving as a control. Toroidal scaffolds formed out of the lamellar, and porous silk materials were used to generate structures with an outer diameter of 8 mm, inner diameter of 3.5 mm, and a height of 3 mm (the interlamellar distance in the lamellar scaffold was 150-250 μm, and the average pore sizes in the porous scaffolds were 100-250 μm). The scaffolds were seeded with porcine AF cells to form AF tissue, whereas porcine chondrocytes were encapsulated in fibrin/HA hydrogels for the NP tissue and embedded in the center of the toroidal disk. Histology, biochemical assays, and gene expression indicated that the lamellar scaffolds supported AF-like tissue over 2 weeks. Porcine chondrocytes formed the NP phenotype within the hydrogel after 4 weeks of culture with the AF tissue that had been previously cultured for 2 weeks, for a total of 6 weeks of cultivation. This biphasic scaffold simulating in combination of both AF and NP tissues was effective in the formation of the total IVD in vitro.

FIG. 1.

(a) Experimental scheme: nucleus pulposus (NP) tissue differentiation for additional 4 weeks (annulus fibrosus [AF]-NP 4 weeks) after AF tissue induction for 2 weeks (AF 2 weeks), (b) biphasic intervertebral disk (IVD) structure (silk scaffold combined with fibrin/hyaluronic acid [HA] gel) using porcine-derived AF cells and chondrocytes. Color images available online at www.liebertonline.com/tea

FIG. 2.

SEM images of lamellar and porous scaffolds (a) lamellar scaffolds (b) porcine AF cells on lamellar at AF-2 weeks, (d) porous scaffolds, (e) porcine AF cells on porous scaffolds at AF-2 weeks. H&E staining for cells: (c) lamellar scaffolds, (f) porous scaffolds. Scale bars=200 μm. Arrows indicate cells on the scaffolds, and S indicates silk. Color images available online at www.liebertonline.com/tea

FIG. 3.

Live/dead staining of cells in biphasic structures of both silk scaffolds (porcine AF cells) and fibrin/HA gel (porcine chondrocytes) at AF-NP 2 weeks: (a) fibrin/HA gel in the center of lamellar AF, (b) lamellar AF scaffold, (c) fibrin/HA gel in the center of porous AF scaffold, and (d) porous AF scaffold. Scale bars=300 μm. Color images available online at www.liebertonline.com/tea

FIG. 4.

Histological and immunohistochemical staining of biphasic structures at AF-NP 4 weeks. Lamellar scaffold: (a) alcian blue staining of entire structure, (b) hematoxylin and eosin (H&E) staining of merged region between AF and NP, (c) type I collagen staining of lamellar scaffolds, (d) type II collagen staining of fibrin/HA gel. Porous scaffold: (e) alcian blue staining of entire structure, (f) H&E staining of merged area between AF and NP, (g) type I collagen staining of porous scaffold, (h) type II collagen staining of fibrin/HA gel. Extracellular matrix stained positive for type I and II collagen with brown color (solid arrows). Dash arrows indicate integration area between silk scaffolds for AF and gel for NP. (A, E) scale bars=3 mm, (b, c, d, f, g, h) scale bars=100 μm. The dotted rectangle furthest to the left is shown in the bottom left (c and g). The other dotted rectangle is shown in the bottom right (d and h) and the solid rectangle is shown in top right (b and f). Color images available online at www.liebertonline.com/tea

FIG. 5.

Chemical analysis of silk scaffolds of AF region at AF-NP 4 weeks: (a) DNA content, (b) GAGs, (c) total collagen content per construct. Data shown as mean±standard deviation, 4 samples (*p<0.05, **p<0.01, and ***p<0.001). GAGs, glycosaminoglycans. Color images available online at www.liebertonline.com/tea

FIG. 6.

Chemical analysis of fibrin/HA gel of NP region at AF-NP 4 weeks: (a) DNA content, (b) GAGs, (c) total collagen content per construct. Statistically significant differences, n=4 (*p<0.05). Color images available online at www.liebertonline.com/tea

FIG. 7.

Transcript levels related to IVD tissue differentiation markers at AF-NP 4 weeks: (a) aggrecan, (b) collagen type Iα1 (Col Iα1), and (c) collagen type IIα1 (Col IIα1). Data quantified by real-time PCR and normalized to GAPDH within the linear range of amplification. Data are shown as mean±standard deviation from N=4, *p<0.05 and **p<0.01.