Development of a bovine decellularized extracellular matrix-biomaterial for nucleus pulposus regeneration

Abstract

Painful intervertebral disc (IVD) degeneration is a common cause for spinal surgery. There is a clinical need to develop injectable biomaterials capable of promoting IVD regeneration, yet many available biomaterials do not mimic the native extracellular matrix (ECM) or promote matrix production. This study aimed to develop a decellularized injectable bovine ECM material that maintains structural and compositional features of native tissue and promotes nucleus pulposus (NP) cell (NPC) and mesenchymal stem cell (MSC) adaption. Injectable decellularized ECM constructs were created using 3 NP tissue decellularization methods (con.A: sodium deoxycholate, con.B: sodium deoxycholate & sodium dodecyl sulfate, con.C: sodium deoxycholate, sodium dodecyl sulfate & TritonX-100) and evaluated for protein, microstructure, and for cell adaptation in 21 day human NPC and MSC culture experiments. Con.A was most efficient at DNA depletion, preserved best collagen microstructure and content, and maintained the highest glycosaminoglycan (GAG) content. NPCs in decellularized constructs of con.A&B demonstrated newly synthesized GAG production, which was apparent from “halos” of GAG staining surrounding seeded NPCs. Con.A also promoted MSC adaption with high cell viability and ECM production. The injectable decellularized NP biomaterial that used sodium deoxycholate without additional decellularization steps maintained native NP tissue structure and composition closest to natural ECM and promoted cellular adaptation of NP cells and MSCs. This natural decellularized biomaterial warrants further investigation for its potential as an injectable cell seeded supplement to augment NP replacement biomaterials and deliver NPCs or MSCs.

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

“…An optimized decellularization process for nucleus pulposus needs to balance the removal of cells with the maintenance of sGAGs. The sGAGs are essential in maintaining the osmotic pressure of the nucleus pulposus [8], and they have the potential to play a role in driving cell phenotype [45][46][47]50,58] and preventing innervation [59][60][61][62]. The gentle decellularization protocol used in our laboratory for nerve [39,43,63] and lung tissue [38] was adapted for use in the nucleus pulposus by shortening the duration of wash cycles substantially.…”

“…Substantial work has been performed in other tissue types to create injectable in situ gelling decellularized matrices [37,51,54], but no work exists in the IVD. To date, the only injectable decellularized nucleus pulposus was developed by IllienJünger et al [47]. However, this matrix was created by grinding the tissue to create small particles that were injected through a 25 gauge needle.…”

“…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.…”

Correlation between T2* (T2 star) relaxation time and cervical intervertebral disc degeneration

“…[1–3] Early signs of disc degeneration are manifested by biochemical changes, including proteoglycan loss and decreased osmotic pressure and hydration. [4,5] In the later stages of disc degeneration, morphological changes occur, including a loss of disc height, disc herniation, annular tears, and radial bulging. [6] …”

Critical aspects and challenges for intervertebral disc repair and regeneration—Harnessing advances in tissue engineering

“…An attractive approach consists in the generation of decellularized matrices from healthy NP. Decellularization processes obtain biomaterials that represent the native tissue microstructure and biochemistry, supporting cellular adaptation . Further preclinical studies are warranted to compare such matrices with other synthetic or natural biomaterials.…”