Abstract
Introduction
Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc diseases. Potential alterations of immunogenic properties along intervertebral disc (IVD) cell isolation and expansion and in three-dimensional (3D) culture with various biomaterials, however, have been scarcely addressed in the literature. Therefore, our aim was to evaluate the immunogenic properties of mild and severely degenerated nucleus pulposus (NP) cells with regard to cell surface markers and cocultures with autologous or allogenic peripheral blood mononuclear cells (PBMC) as well as changes of theses immunogenic properties after 3D culture in a polymer-based porous biomaterial.
Materials and Methods
Ten patients with cervical disc herniations, five with severe and five with mild grades of IVD degeneration according to the Miyazaki MRI classification, were operated, and tissue from the NP compartment was carefully selected. NP cells were isolated, expanded, and cultured in 3D fibrin/poly(lactic-coglycolic) acid transplants for 21 days. NP cells were evaluated for induction of immune responses in coculture with PBMC from the same donor or from an unrelated healthy volunteer, using a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay. Surface marker detection and evaluation of cytokines was performed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS) analysis.
Results
NP cells from donors with severe degeneration tended to have higher expression of the adhesion molecules ICAM and VCAM, as well as Fas ligand, though only VCAM was significantly increased. All cells expressed HLA-ABC and CD95 (Fas), but lacked expression of HLA-DR and CD24. NP cells after monolayer culture did not provoke a significant proliferation of T cells, NK cells, or B cells from either the same donor or from allogenic PBMC. Proliferating capacity was verified with the mitogenic stimulator ConA. When NP cells were incorporated into a 3D matrix, the immune cell proliferation was provoked to a greater extent than for NP cells preseeded as a monolayer. 3D specimens containing NP cells from patients with severe degeneration tended to provoke a larger response, especially for CD3+CD8+ and CD3+CD4+ T cells, than those with mild degeneration, though these differences were not significant. In general, the cytokine release was low or undetectable, especially for the inflammatory cytokines TNF-α and IFN-gamma.
Conclusion
NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, NK cells, or B cells, not only with donor PBMC but also with allogenic PBMC. In conjunction with low inflammatory cytokine expression, a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D culture with a fibrin/poly(lactic-coglycolic) acid matrix, however, we found higher 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 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.
None declared