Comparison of Decellularization Protocols for Preparing a Decellularized Porcine Annulus Fibrosus Scaffold

TLDR

Tissue‑specific extracellular matrix is essential for tissue regeneration and repair. The study aimed to identify the optimal decellularization method for creating a suitable annular fibrosus scaffold for tissue engineering. Three decellularization protocols—Triton X‑100, SDS, and trypsin—were applied to annular fibrosus tissue, and the resulting cell removal, matrix preservation, microstructure, and mechanical properties were evaluated. All three protocols achieved decellularization, but SDS or trypsin disrupted the annular fibrosus structure, whereas Triton X‑100 preserved collagen, maintained higher glycosaminoglycan content, retained tensile mechanical properties, and showed no cytotoxicity, indicating it is a suitable scaffold for annular fibrosus tissue engineering.

Abstract

Tissue-specific extracellular matrix plays an important role in promoting tissue regeneration and repair. We hypothesized that decellularized annular fibrosus matrix may be an appropriate scaffold for annular fibrosus tissue engineering. We aimed to determine the optimal decellularization method suitable for annular fibrosus. Annular fibrosus tissue was treated with 3 different protocols with Triton X-100, sodium dodecyl sulfate (SDS) and trypsin. After the decellularization process, we examined cell removal and preservation of the matrix components, microstructure and mechanical function with the treatments to determine which method is more efficient. All 3 protocols achieved decellularization; however, SDS or trypsin disturbed the structure of the annular fibrosus. All protocols maintained collagen content, but glycosaminoglycan content was lost to different degrees, with the highest content with TritonX-100 treatment. Furthermore, SDS decreased the tensile mechanical property of annular fibrosus as compared with the other 2 protocols. MTT assay revealed that the decellularized annular fibrosus was not cytotoxic. Annular fibrosus cells seeded into the scaffold showed good viability. The Triton X-100–treated annular fibrosus retained major extracellular matrix components after thorough cell removal and preserved the concentric lamellar structure and tensile mechanical properties. As well, it possessed favorable biocompatibility, so it may be a suitable candidate as a scaffold for annular fibrosus tissue engineering.