Regenerative and Immunogenic Characteristics of Cultured Nucleus Pulposus Cells from Human Cervical Intervertebral Discs

Abstract

Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD) diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP) cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC) including changes in their immunogenic properties after 3-dimensional (3D)-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic) acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR) showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK) cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS), a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T-cell activity acting by Fas ligand-induced apoptosis.

The Immune Privilege of the Intervertebral Disc: Implications for Intervertebral Disc Degeneration Treatment

“…Moreover, inflammatory factors were found to have increased expression in the study of Takada et al, who found IVD autografts induced TNFα, IL-6 and IL-8; as well as cyclooxygenase 2 up-regulation and macrophage infiltration in sciatica [46]. By co-culture of autologous or allogeneic peripheral blood mononuclear cells and NP cells, Stich et al found elevated immune cell proliferation levels in 3Dcultures than 2D-cultures, and a general trend to higher responses for NP cells from severely degenerated IVD tissue [47], indicating that autoimmune response could vary depending on different cell cultures and degeneration degrees. More recently, Silva et al established a model of IVD organ culture, found that human macrophages could be polarized toward a more pro-inflammatory profile by degenerated IVD tissue, and interfere with IVD ECM remodeling by downregulating aggrecan and collagen II gene expression in the presence of IL-1β [48].…”

Are specific gene expressions of extracellular matrix and nucleus pulposus affected by primer cell cultures prepared from intact or degenerative intervertebral disc tissues?

“…Stich et al indicated that cell-based regenerative approaches could be advised as a primary or adjuvant procedure in order to prevent degeneration in IVD treatment (24). They tested middle-and upper-level degenerated cervical NP cells in terms of cell renewal, proliferation, and differentiation.…”

Transplantation of allogenic nucleus pulposus cells attenuates intervertebral disc degeneration by inhibiting apoptosis and increasing migration

“…However, Okuma et al ( 9 ) reported that the reinsertion of stimulated NPCs delayed IVD degeneration. NPCs were also used to build 3D tissue engineering complexes to repair IVD ( 28 ). Ganey et al ( 18 ) demonstrated that DC transplantation was a viable treatment for degenerated or damaged IVD.…”