Silk-based multilayered angle-ply annulus fibrosus construct to recapitulate form and function of the intervertebral disc

Abstract

Recapitulation of the form and function of complex tissue organization using appropriate biomaterials impacts success in tissue engineering endeavors. The annulus fibrosus (AF) represents a complex, multilamellar, hierarchical structure consisting of collagen, proteoglycans, and elastic fibers. To mimic the intricacy of AF anatomy, a silk protein-based multilayered, disc-like angle-ply construct was fabricated, consisting of concentric layers of lamellar sheets. Scanning electron microscopy and fluorescence image analysis revealed cross-aligned and lamellar characteristics of the construct, mimicking the native hierarchical architecture of the AF. Induction of secondary structure in the silk constructs was confirmed by infrared spectroscopy and X-ray diffraction. The constructs showed a compressive modulus of 499.18 ± 86.45 kPa. Constructs seeded with porcine AF cells and human mesenchymal stem cells (hMSCs) showed ∼2.2-fold and ∼1.7-fold increases in proliferation on day 14, respectively, compared with initial seeding. Biochemical analysis, histology, and immunohistochemistry results showed the deposition of AF-specific extracellular matrix (sulfated glycosaminoglycan and collagen type I), indicating a favorable environment for both cell types, which was further validated by the expression of AF tissue-specific genes. The constructs seeded with porcine AF cells showed ∼11-, ∼5.1-, and ∼6.7-fold increases in col I, sox 9, and aggrecan genes, respectively. The differentiation of hMSCs to AF-like tissue was evident from the enhanced expression of the AF-specific genes. Overall, the constructs supported cell proliferation, differentiation, and ECM deposition resulting in AF-like tissue features based on ECM deposition and morphology, indicating potential for future studies related to intervertebral disc replacement therapy.

In Situ Forming Injectable Silk Fibroin Hydrogel Promotes Skin Regeneration in Full Thickness Burn Wounds

“…The peaks of BmSF, AaSF, and blend matrices were found to be distinct. Sharp peaks at 2θ = 12° and 21° were observed for AaSF; whereas a single broad peak around 20° was observed for BmSF depicting amorphous nature of silk proteins . In contrast, SF hydrogel matrix made by blending AaSF and BmSF demonstrated broad peak at 21° with higher intensity.…”

Replication of annulus fibrosus through fabrication and characterization of polyurethane and cellulose nanocrystal composite scaffolds

“…The majority of these scaffolds are comprised of aligned polymer fibers with a type I collagen rich extracellular matrix for the AF and an alginate or type II collagen hydrogel for the NP, with varying polymers and embedded biological components throughout different studies. 26,27,78,79 These scaffolds, however, focused more on the biological aspect of the system rather than the manipulation of the mechanical properties. Therefore, none of these tissue engineering options demonstrate the tunability shown in this study through the variability of CNC concentration and amount of rings to manipulate the mechanical properties of the scaffolds.…”

“…The tunability of the CNC concentration also leads to the possibility of gradient scaffolds, such as making the outer rings stiffer and inner rings softer. 27,78,79 Although the composite materials in this study do not contain any biological components, an argument can be made for the advantages of the nonbiological scaffold. Most of the current research approaches rely on the biological components to yield the properties necessary to replace the natural AF and IVD, however, these biological components are what lead to IVD degeneration.…”

Fabrication of a novel whole tissue-engineered intervertebral disc for intervertebral disc regeneration in the porcine lumbar spine

“…Many previous studies have been concentrated on regenerating a specic component of IVD, including NP or AF separately. [29][30][31] Also, the regenerative IVD cartilage endplate capable of nutrients and metabolites exchange was prepared in our previous study. 32 However, the separate scaffolds could not emulate the structure and mechanical properties of natural IVD.…”