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

Abstract

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

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

“…There was also no significant difference in collagen content between native and decellularized AF. Our present data showed that 5.6% of GAG was lost during processing, which was an improvement over our previous protocol, which resulted in a 15.9% loss of GAG (Wu et al 2014 ).…”

“…One tissue engineering strategy for developing biomaterials is to decellularize xenogeneic tissues by removing immunogenic cells. This method has been successfully used to develop biomaterials for use in cartilage, the meniscus, ligaments, and tendons, with impressive results (Gilbert et al 2006 , 2009 ; Stone et al 2007 ; Wu et al 2014 ; Xu et al 2014 ). Xenogeneic and allogeneic cellular antigens are recognized as foreign by the host, thereby inducing an inflammatory response or an immune-mediated rejection of the tissue.…”

“…We previously reported a preliminary method for decellularizing porcine AF to yield a biological scaffold for use in human AF defect substitution (Wu et al 2014 ). Our method produced a scaffold with low DNA content that retained most of the original biological composition and properties, with some loss of glycosaminoglycan (GAG).…”

Creation of an injectable in situ gelling native extracellular matrix for nucleus pulposus tissue engineering

“…Our laboratory has previously developed gentle decellularization techniques for nerve tissue to preserve essential proteins, proteoglycans, and microstructure [39,43], which would be ideal for the delicate nucleus pulposus. To date, some research has been performed to create decellularization protocols for the nucleus pulposus [44][45][46][47], anulus fibrosus [48,49], and the intact IVD [50]. These matrices have shown promise as viable scaffolds for IVD cells as well as drivers of stem cell fate [44][45][46][47][48][49][50], although they are not injectable or in situ gelling.…”

Construction Strategy and Progress of Whole Intervertebral Disc Tissue Engineering

“…The AF can be divided into inner and outer layers, the outer layer being mainly composed of type I collagen, whereas the inner AF is mainly type II collagen. From outer to inner AF, the content of water and proteoglycans increases, whereas the content of type I collagen gradually decreases . The NP, which is located in the central region of the IVD and has a jelly‐like structure, is rich in proteoglycan, type II collagen and water .…”