Optimized decellularization protocol including α-Gal epitope reduction for fabrication of an acellular porcine annulus fibrosus scaffold

Abstract

Recent advances in tissue engineering have led to potential new strategies, especially decellularization protocols from natural tissues, for the repair, replacement, and regeneration of intervertebral discs. This study aimed to validate our previously reported method for the decellularization of annulus fibrosus (AF) tissue and to quantify potentially antigenic α-Gal epitopes in the decellularized tissue. Porcine AF tissue was decellularized using different freeze–thaw temperatures, chemical detergents, and incubation times in order to determine the optimal method for cell removal. The integrity of the decellularized material was determined using biochemical and mechanical tests. The α-Gal epitope was quantified before and after decellularization. Decellularization with freeze–thaw in liquid nitrogen, an ionic detergent (0.1% SDS), and a 24 h incubation period yielded the greatest retention of GAG and collagen relative to DNA reduction when tested as single variables. Combined, these optimal decellularization conditions preserved more GAG while removing the same amount of DNA as the conditions used in our previous study. Components and biomechanical properties of the AF matrix were retained. The decellularized AF scaffold exhibited suitable immune-compatibility, as evidenced by successful in vivo remodeling and a decrease in the α-Gal epitope. Our study defined the optimal conditions for decellularization of porcine AF tissues while preserving the biological composition and mechanical properties of the scaffold. Under these conditions, immunocompatibility was evidenced by successful in vivo remodeling and reduction of the α-Gal epitope in the decellularized material. Decellularized AF scaffolds are potential candidates for clinical applications in spinal surgery.

Optimizing detergent concentration and processing time to balance the decellularization efficiency and properties of bioprosthetic heart valves

“…It has been accepted that the presence of α‐Gal will induce the rejection of porcine heart valves (Naso et al, ; Naso, Gandaglia, Iop, Spina, & Gerosa, ). It showed that the decellularization procedures could diminish the content of antigen (Wu et al, ). An Enzyme‐linked immunosorbent assay (ELISA) based quantitative method was used to assess the number of epitopes of native valves and decellularized valves (Figure ).…”

Annulus fibrosus cell sheets limit disc degeneration in a rat annulus fibrosus injury model

“…Moreover, in vivo AF regeneration studies have resulted in mixed but suboptimal outcomes . Decellularized AF grafts applied in rat annuluotomy‐mediated AF‐defect rat model for AF repair resulted in large scar tissue formation . On the other hand, application of crosslinked collagen constructs applied in a rat needle‐puncture model, showed strongly enhanced IVD features compared to nontreated discs, but still reported atypical AF tissue organization .…”

Decellularization and characterization of a whole intervertebral disk xenograft scaffold

“…The primary objective of decellularizing allogenic or xenogenic tissues for the creation of biomaterial scaffolds is to remove all cells, nuclear material and antigenic epitopes while maintaining critical ECM components, their spatial distribution, relative ratios, and tissue microarchitecture. 22,[28][29][30][31][32][33][34][35][36][37][38] This top-down approach to scaffold development aims to minimize the possibility of immune rejection while yielding a biomimetic scaffold that can promote targeted tissue regeneration. Moreover, this approach offers several advantages compared to building scaffolds from the ground-up including; (1) obtaining a "prefabricated," tissuespecific microarchitecture without employing complex additive manufacturing techniques, which (2) yields scaffolds that provide "built-in" instructional cues to local/seeded cells instructing them to attain tissue-specific phenotypes and contribute to tissue regeneration even in the absence of other exogenous soluble growth and differentiation factors.…”