Establishment of a Cytocompatible Cell-Free Intervertebral Disc Matrix for Chondrogenesis with Human Bone Marrow-Derived Mesenchymal Stromal Cells

Abstract

Tissue-engineered intervertebral discs (IVDs) utilizing decellularized extracellular matrix (ECM) could be an option for the reconstruction of impaired IVDs due to degeneration or injury. The objective of this study was to prepare a cell-free decellularized human IVD scaffold and to compare neotissue formation in response to recellularization with human IVD cells (hIVDCs) or human bone marrow-derived (hBM) mesenchymal stromal cells (MSCs). IVDs were decellularized via freeze-thaw cycles, detergents and trypsin. Histological staining was performed to monitor cell removal and glycosaminoglycan (GAG) removal. The decellularized IVD was preconditioned using bovine serum albumin and fetal bovine serum before its cytocompatibility for dynamically cultured hBM-MSCs (chondrogenically induced or not) and hIVDCs was compared after 14 days. In addition, DNA, total collagen and GAG contents were assessed. The decellularization protocol achieved maximal cell removal, with only few remaining cell nuclei compared with native tissue, and low toxicity. The DNA content was significantly higher in scaffolds seeded with hIVDCs compared with native IVDs, cell-free and hBM-MSC-seeded scaffolds (p < 0.01). The GAG content in the native tissue was significantly higher compared to the others groups except for the scaffolds reseeded with chondrogenically induced hBM-MSCs (p < 0.05). In addition, there was a significantly increased total collagen content in the chondrogenically induced hBM-MSCs group (p < 0.01) compared with the native IVDs, cell-free and hIVDC-seeded scaffolds (p < 0.01); both recolonizing cell types were more evenly distributed on the scaffold surface, but only few cells penetrated the scaffold. The resulting decellularized ECM was cytocompatible and allowed hBM-MSCs/hIVDCs survival and ECM production.

Ethanol‐mediated compaction and cross‐linking enhance mechanical properties and degradation resistance while maintaining cytocompatibility of a nucleus pulposus scaffold

“…This was in agreement with our bovine explant FSU model, for which the ABNP was not able to restore the viscoelastic creep parameters following repair . These mechanical deficiencies were attributed to decellularization‐induced changes that increased ECM porosity and permeability due to free‐swelling, GAG leaching, and tissue damage, which has been observed in other decellularization methodologies for NP tissue …”

“…Decellularized NP therapies have been previously reported, but many are implemented as injectable derivatives of acellular ECM, and have not yet been evaluated for mechanical function postinjection. Decellularization of intact human NP was reported by Huang et al, who demonstrated devitalization of the tissue through the absence of nuclei within lacunae and the preservation of native ECM components . However, quantification of DNA content showed no difference between native and decellularized tissues, which could induce an adverse host reaction in response to residual exogenous DNA, and the decellularized tissue was not mechanically characterized.…”

Decellularization and characterization of a whole intervertebral disk xenograft scaffold

“…22,28–38 This top-down approach to scaffold development aims to minimize the possibility of immune rejection while yielding a biomimetic scaffold that can promote targeted tissue regeneration. Moreover, this approach offers several advantages compared to building scaffolds from the ground-up including; 1) obtaining a ‘pre-fabricated,’ tissue-specific micro-architecture without employing complex additive manufacturing techniques, which 2) yields scaffolds that provide ‘built-in’ instructional cues to local/seeded cells instructing them to attain tissue-specific phenotypes and contribute to tissue regeneration even in the absence of other exogenous soluble growth and differentiation factors.…”

“…The primary objective of decellularizing allogenic or xenogenic tissues for the creation of biomaterial scaffolds is to remove all cells, nuclear material and antigenic epitopes while maintaining critical ECM components, their spatial distribution, relative ratios, and tissue microarchitecture. 22,[28][29][30][31][32][33][34][35][36][37][38] This top-down approach to scaffold development aims to minimize the possibility of immune rejection while yielding a biomimetic scaffold that can promote targeted tissue regeneration. Moreover, this approach offers several advantages compared to building scaffolds from the ground-up including; (1) obtaining a "prefabricated," tissuespecific microarchitecture without employing complex additive manufacturing techniques, which (2) yields scaffolds that provide "built-in" instructional cues to local/seeded cells instructing them to attain tissue-specific phenotypes and contribute to tissue regeneration even in the absence of other exogenous soluble growth and differentiation factors.…”

Ethanol-Mediated Compaction and Crosslinking Enhance Mechanical Properties and Degradation Resistance While Maintaining Cytocompatibility of a Nucleus Pulposus Scaffold

“…Decellularization of intact human NP was reported by Huang et al, who demonstrated devitalization of the tissue through the absence of nuclei within lacunae and the preservation of native ECM components. 33 However, quantification of DNA content showed no difference between native and decellularized tissues, which could induce an adverse host reaction in response to residual exogenous DNA, 34,35 and the decellularized tissue was not mechanically characterized.…”