Optimized decellularization protocol including α-Gal epitope reduction for fabrication of an acellular porcine annulus fibrosus scaffold.
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.
Institute of Biomedical Engineering, College of Engineering, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan.|Department of Orthopedics, Shuang Ho Hospital, Taipei Medical University, Taipei, 23561, Taiwan.|Department of Orthopaedics, School of Medicine, College of Medicine, Taipei Medical University, Taipei, 11031, Taiwan.
Lien-Chen
LC
Wu
Department of Orthopedics, Taipei Medical University Hospital, Taipei, 110, Taiwan.|Department of Orthopaedics, School of Medicine, College of Medicine, Taipei Medical University, Taipei, 11031, Taiwan.
Yi-Jie
YJ
Kuo
Institute of Biomedical Engineering, College of Engineering, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan.
Fu-Wen
FW
Sun
Department of Orthopedics, Shuang Ho Hospital, Taipei Medical University, Taipei, 23561, Taiwan.
Chia-Hsien
CH
Chen
Department of Orthopedics, Shuang Ho Hospital, Taipei Medical University, Taipei, 23561, Taiwan.|Department of Orthopaedics, School of Medicine, College of Medicine, Taipei Medical University, Taipei, 11031, Taiwan.
Chang-Jung
CJ
Chiang
Department of Orthopedics, Shuang Ho Hospital, Taipei Medical University, Taipei, 23561, Taiwan.|Department of Orthopaedics, School of Medicine, College of Medicine, Taipei Medical University, Taipei, 11031, Taiwan.
Pei-Wei
PW
Weng
Department of Orthopedics, Shuang Ho Hospital, Taipei Medical University, Taipei, 23561, Taiwan.|Department of Orthopaedics, School of Medicine, College of Medicine, Taipei Medical University, Taipei, 11031, Taiwan.
Yang-Hwei
YH
Tsuang
Institute of Biomedical Engineering, College of Engineering, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan. yyhuang@ntu.edu.tw.
Yi-You
YY
Huang
D000818:Animals; D000070616:Annulus Fibrosus / Q000737:chemistry* / Q000166:cytology; D001696:Biomechanical Phenomena; D055119:Elastic Modulus; D000939:Epitopes / Q000032:analysis; D005690:Galactose / Q000032:analysis; D051379:Mice; D041681:NIH 3T3 Cells; D013552:Swine; D023822:Tissue Engineering / Q000379:methods; D054457:Tissue Scaffolds / Q000737:chemistry*
D000939:Epitopes; D005690:Galactose
Annulus fibrosus; Decellularization; Intervertebral disc; Tissue engineering; α-Gal epitope