Fabrication and properties of acellular porcine anulus fibrosus for tissue engineering in spine surgery

Journal of Orthopaedic Surgery and Research

Yi-You Huang²  Jui-Sheng Sun³  Yang-Hwei Tsuang¹  Zen-Hao Liu²  Chang-Jung Chiang¹  Lien-Chen Wu¹ 

[1] Department of Orthopedics, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan;Institute of Biomedical Engineering, College of Engineering, College of Medicine, National Taiwan University, No.1, Sec.1, Jen-Ai Road, Taipei, Taiwan;Department of Orthopaedics, National Taiwan University Hospital HsinChu Branch, HsinChu, Taiwan

关键词: Tissue engineering;    Intervertebral disc;    Disc degeneration;    Acellular;    Decellularization;    Anulus fibrosus;   

Others  :  1144321
DOI  :  10.1186/s13018-014-0118-z

 received in 2014-06-18, accepted in 2014-11-05,  发布年份 2014

【 摘 要 】

Background

Over the last few years, new treatments for a damaged intervertebral disc (IVD) have included strategies to repair, replace, or regenerate the degenerative disc. However, these techniques are likely to have limited success, due to insufficiently effective means to address the damaged anulus fibrosus (AF). Here, we try to develop a bioprocess method for decellularization of the xenogeneic AF tissue, with a view to developing a scaffold as a potential candidate for clinical application in spinal surgery.

Methods

Porcine AFs were decellularized using freeze-thaw cycles, followed by various combined treatments with 0.1% sodium dodecyl sulfate (SDS) and nucleases.

Results

Hematoxylin and eosin (H & E) staining showed that decellularization was achieved through the decellularization protocols. Biochemical analyses revealed 86% reduction in DNA, but only 15.9% reduction in glycosaminoglycan (GAG) content, with no significant difference in the hydroxyproline content. There was no appreciable cytotoxicity of the acellular AF. Biomechanical testing of the acellular AF found no significant decline in stiffness or Young’s modulus.

Conclusions

Porcine AF tissues were effectively decellularized with the preservation of biologic composition and mechanical properties. These results demonstrate that acellular AF scaffolds would be a potential candidate for clinical application in spinal surgery.

【 授权许可】

   
2014 Wu et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Chan SC, Gantenbein-Ritter B: Intervertebral disc regeneration or repair with biomaterials and stem cell therapy–feasible or fiction? Swiss Med Wkly 2012, 142:w13598.
• [2]Bron JL, Helder MN, Meisel HJ, Van Royen BJ, Smit TH: Repair, regenerative and supportive therapies of the annulus fibrosus: achievements and challenges. Eur Spine J 2009, 18(3):301-313.
• [3]Mwale F, Masuda K, Pichika R, Epure LM, Yoshikawa T, Hemmad A, Roughley PJ, Antoniou J: The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration. Arthritis Res Ther 2011, 13(4):R120. BioMed Central Full Text
• [4]Li Jin ALS, Xudong L: The challenge and advancement of annulus fibrosus tissue engineering. Eur Spine J 2013, 22(5):1090-1100.
• [5]Guterl CC, See EY, Blanquer SBG, Pandit A, Ferguson SJ, Benneker LM, Grijpma DW, Sakai D, Eglin D, Alini M, Iatridis JC, Grad S: Challenges and strategies in the repair of ruptured annulus fibrosus. Eur Cell Mater 2013, 25:1-21.
• [6]Gilbert TW, Sellaro TL, Badylak SF: Decellularization of tissues and organs. Biomaterials 2006, 27(19):3675-3683.
• [7]Gilbert TW, Freund JM, Badylak SF: Quantification of DNA in biologic scaffold materials. J Surg Res 2009, 152(1):135-139.
• [8]Liu Yang RAK, Godfrey Chang J, Paul S: Polar surface chemistry of nanofibrous polyurethane scaffold affects annulus fibrosus cell attachment and early matrix accumulation. J Biomed Mater Res A 2009, 15(91(4)):1089-1099.
• [9]Mercuri JJ, Gill SS, Simionescu DT: Novel tissue-derived biomimetic scaffold for regenerating the human nucleus pulposus. J Biomed Mater Res A 2011, 96A(2):422-435.
• [10]Schek RM, Michalek AJ, Iatridis JC: Genipin-crosslinked fibrin hydrogels as a potential adhesive to augment intervertebral disc annulus repair. Eur Cell Mater 2011, 21:373-383.
• [11]Chiang CJ, Cheng CK, Sun JS, Liao CJ, Wang YH, Tsuang YH: The effect of a new anular repair after discectomy in intervertebral disc degeneration: an experimental study using a porcine spine model. Spine 2011, 36(10):761-769.
• [12]Ahlgren BD, Lui W, Herkowitz HN, Panjabi MM, Guiboux JP: Effect of anular repair on the healing strength of the intervertebral disc: a sheep model. Spine 2000, 25(17):2165-2170.
• [13]Booth C, Korossis SA, Wilcox HE, Watterson KG, Kearney JN, Fisher J, Ingham E: Tissue engineering of cardiac valve prostheses I: development and histological characterization of an acellular porcine scaffold. J Heart Valve Dis 2002, 11(4):457-462.
• [14]Stapleton TW, Ingram J, Katta J, Knight R, Korossis S, Fisher J, Ingham E: Development and characterization of an acellular porcine medial meniscus for use in tissue engineering. Tissue Eng Part A 2008, 14(4):505-518.
• [15]Farndale RWBD, Barrett AJ: Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 1986, 883(2):173-177.
• [16]Zhang L, Dong Y, Cheng J, Du J: Role of integrin-beta3 protein in macrophage polarization and regeneration of injured muscle. J Biol Chem 2012, 287(9):6177-6186.
• [17]Rabelo-Goncalves E, Roesler B, Guardia AC, Milan A, Hara N, Escanhoela C, Almeida J, Boin I, Zeitune JM: Evaluation of five DNA extraction methods for detection of H. pylori in formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with hepatocellular carcinoma. Pathol Res Pract 2014, 210(3):142-146.
• [18]Mitchell R, Ladd SJL, Joel DS, Anthony A, Yoo JJ: Co-electrospun dual scaffolding system with potential for muscle-tendon junction tissue engineering. Biomaterials 2011, 32(6):1549-1559.
• [19]Penn D, Willet TL, Glazebrook M, Snow M, Stanish WD: Is there significant variation in the material properties of four different allografts implanted for ACL reconstruction. Knee Surg Sport Tr A 2009, 17(3):260-265.
• [20]Woo SL, Hollis JM, Adams DJ, Lyon RM, Takai S: Tensile properties of the human femur-anterior cruciate ligament-tibia complex. The effects of specimen age and orientation. Am J Sports Med 1991, 19(3):217-225.
• [21]Rieder E, Kasimir MT, Silberhumer G, Seebacher G, Wolner E, Simon P, Weigel G: Decellularization protocols of porcine heart valves differ importantly in efficiency of cell removal and susceptibility of the matrix to recellularization with human vascular cells. J Thorac Cardiovasc Surg 2004, 127(2):399-405.
• [22]Woods T, Gratzer PF: Effectiveness of three extraction techniques in the development of a decellularized bone-anterior cruciate ligament-bone graft. Biomaterials 2005, 26(35):7339-7349.
• [23]Huang H, Zhang J, Sun K, Zhang X, Tian S: Effects of repetitive multiple freeze-thaw cycles on the biomechanical properties of human flexor digitorum superficialis and flexor pollicis longus tendons. Clin Biomech (Bristol, Avon) 2011, 26(4):419-423.
• [24]Hongo M, Gay RE, Hsu JT, Zhao KD, Ilharreborde B, Berglund LJ, An KN: Effect of multiple freeze-thaw cycles on intervertebral dynamic motion characteristics in the porcine lumbar spine. J Biomech 2008, 41(4):916-920.
• [25]Dahl SL, Koh J, Prabhakar V, Niklason LE: Decellularized native and engineered arterial scaffolds for transplantation. Cell Transplant 2003, 12(6):659-666.
• [26]Suto K, Urabe K, Naruse K, Uchida K, Matsuura T, Mikuni-Takagaki Y, Nemoto N, Kamiya K, Itoman M: Repeated freeze-thaw cycles reduce the survival rate of osteocytes in bone-tendon constructs without affecting the mechanical properties of tendons. Cell Tissue Bank 2012, 13(1):71-80.
• [27]McPherson TB, Liang H, Record RD, Badylak SF: Galalpha(1,3)Gal epitope in porcine small intestinal submucosa. Transpl Immunol 2002, 10(1):15-24.
• [28]Raeder RH, Badylak SF, Sheehan C, Kallakury B, Metzger DW: Natural anti-galactose alpha1,3 galactose antibodies delay, but do not prevent the acceptance of extracellular matrix xenografts. Transpl Immunol 2002, 10(1):15-24.
• [29]Daly KA, Stewart-Akers AM, Hara H, Ezzelarab M, Long C, Cordero K, Johnson SA, Ayares D, Cooper DK, Badylak SF: Effect of the alphaGal epitope on the response to small intestinal submucosa extracellular matrix in a nonhuman primate model. Tissue Eng Part A 2009, 15(12):3877-3888.
• [30]Zheng MH, Chen J, Kirilak Y, Willers C, Xu J, Wood D: Porcine small intestine submucosa (SIS) is not an acellular collagenous matrix and contains porcine DNA: possible implications in human implantation. J Biomed Mater Res B Appl Biomater 2005, 73(1):61-67.
• [31]Badylak SF, Gilbert TW: Immune response to biologic scaffold materials. Semin Immunol 2008, 20(2):109-116.